Process for preparing modulators of cystic fibrosis transmembrane conductance regulator

ABSTRACT

The present invention relates to processes for preparing solid state forms of N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1, 4-dihydroquinoline-3-carboxamide, including Compound 1 Form A, Compound 1 Form A-HCl, Compound 1 Form B, and Compound 1 Form B-HCl, any combination of these forms, pharmaceutical compositions thereof, and methods of treatment therewith.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional application U.S. Ser.No. 61/254,634, filed on Oct. 23, 2009. The contents of which isincorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a process for making modulators ofcystic fibrosis transmembrane conductance regulator (“CFTR”).

BACKGROUND

Cystic fibrosis (CF) is a recessive genetic disease that affectsapproximately 30,000 children and adults in the United States andapproximately 30,000 children and adults in Europe. Despite progress inthe treatment of CF, there is no cure.

CF is caused by mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) gene that encodes an epithelial chlorideion channel responsible for aiding in the regulation of salt and waterabsorption and secretion in various tissues. Small molecule drugs, knownas potentiators that increase the probability of CFTR channel openingrepresent one potential therapeutic strategy to treat CF.

Specifically, CFTR is a cAMP/ATP-mediated anion channel that isexpressed in a variety of cell types, including absorptive and secretoryepithelia cells, where it regulates anion flux across the membrane, aswell as the activity of other ion channels and proteins. In epitheliacells, normal functioning of CFTR is critical for the maintenance ofelectrolyte transport throughout the body, including respiratory anddigestive tissue. CFTR is composed of approximately 1480 amino acidsthat encode a protein made up of a tandem repeat of transmembranedomains, each containing six transmembrane helices and a nucleotidebinding domain. The two transmembrane domains are linked by a large,polar, regulatory (R)-domain with multiple phosphorylation sites thatregulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in cysticfibrosis (“CF”), the most common fatal genetic disease in humans. Cysticfibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with CF, mutations in CFTR endogenously expressed inrespiratory epithelia leads to reduced apical anion secretion causing animbalance in ion and fluid transport. The resulting decrease in aniontransport contributes to enhanced mucus accumulation in the lung and theaccompanying microbial infections that ultimately cause death in CFpatients. In addition to respiratory disease, CF patients typicallysuffer from gastrointestinal problems and pancreatic insufficiency that,if left untreated, results in death. In addition, the majority of maleswith cystic fibrosis are infertile and fertility is decreased amongfemales with cystic fibrosis. In contrast to the severe effects of twocopies of the CF associated gene, individuals with a single copy of theCF associated gene exhibit increased resistance to cholera and todehydration resulting from diarrhea—perhaps explaining the relativelyhigh frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, >1000 disease causingmutations in the CF gene have been identified(www.genet.sickkids.on.ca/cftr/app). The most prevalent mutation is adeletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na+ channel,ENaC, Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺-K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺-K⁺-ATPase pumpand Cl⁻ ion channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺-K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases.

Accordingly, there is a need for potent and selective CFTR potentiatorsof wild-type and mutant forms of human CFTR. These mutant CFTR formsinclude, but are not limited to, ΔF508del, G551D, R117H, 2789+5G->A.

There is also a need for modulators of CFTR activity, and compositionsthereof, which can be used to modulate the activity of the CFTR in thecell membrane of a mammal.

There is a need for methods of treating diseases caused by mutation inCFTR using such modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivocell membrane of a mammal.

In addition, there is a need for stable solid forms of said compoundthat can be used readily in pharmaceutical compositions suitable for useas therapeutics.

SUMMARY OF THE INVENTION

The present invention relates to processes for the synthesis ofcompounds useful as modulators of CFTR.

In one aspect, the invention provides A process for making a crystallineform of Compound 1:

comprising:(a) reacting Compound 2 with Compound 3 in the presence of a couplingagent:

wherein the coupling agent is selected from the group consisting of2-chloro-1,3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU,2-chloro-4,6-dimethoxy-1,3,5-triazine, HATU, HOBT/EDC, and T3P®.

Compounds of Formula I and pharmaceutically acceptable compositionsthereof are useful for treating or lessening the severity of a varietyof diseases, disorders, or conditions, including, but not limited to,cystic fibrosis, asthma, smoke induced COPD, chronic bronchitis,rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency,male infertility caused by congenital bilateral absence of the vasdeferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,allergic bronchopulmonary aspergillosis (ABPA), liver disease,hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington's, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease, Osteoporosis, Osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, and PrimaryCiliary Dyskinesia (PCD), a term for inherited disorders of thestructure and/or function of cilia, including PCD with situs inversus(also known as Kartagener syndrome), PCD without situs inversus andciliary aplasia.

In one aspect, Compound 1 is in a crystalline form referred to as FormA.

In another aspect, Compound 1 is in a crystalline form referred to asForm B.

In a further aspect, Compound 1 is in a crystalline form referred to asForm A-HCl.

In an additional aspect, Compound 1 is in a crystalline form referred toas Form B-HCl.

Processes described herein can be used to prepare the compositions ofthis invention comprising Form A, Form A-HCl, Form B, Form B-HCl, or anycombination of these forms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray powder diffraction pattern of a representative sampleof Compound 1 Form A.

FIG. 2 is an FTIR spectrum of a representative sample of Compound 1 FormA.

FIG. 3 is an illustration of the conformational structure of Compound 1Form A based on single X-ray analysis.

FIG. 4 is an X-ray powder diffraction pattern of an exemplary sample ofCompound 1 Form A-HCl.

FIG. 5 is the DSC curve for a representative sample of Compound 1 FormA-HCl.

FIG. 6 is a curve generated by thermogravimetric analysis of arepresentative sample of Compound 1 Form A-HCl that presents sampleweight as a function of temperature.

FIG. 7 is an FTIR spectrum of a representative sample of Compound 1 FormA-HCl.

FIG. 8 is a solid phase ¹³C NMR spectrum of a representative sample ofCompound 1 Form A-HCl.

FIG. 9 is solid phase ¹⁹F NMR spectrum of a representative sample ofCompound 1 Form A-HCl.

FIG. 10A is an X-ray powder diffraction pattern for a representativesample of Compound 1 Form B, recorded with instrument 1.

FIG. 10B is an X-ray powder diffraction pattern for a representativesample of Compound 1 Form B, recorded with instrument 2.

FIG. 11 is the DSC curve for a representative sample of Compound 1 FormB.

FIG. 12 is a curve generated by thermogravimetric analysis of arepresentative sample of Compound 1 Form B that presents sample weightas a function of temperature.

FIG. 13 is an FTIR spectrum of a representative sample of Compound 1Form B.

FIG. 14 is a solid phase ¹³C NMR spectrum of a representative sample ofCompound 1 Form B.

FIG. 15 is solid phase ¹⁹F NMR spectrum of a representative sample ofCompound 1 Form B.

FIG. 16 is an X-ray powder diffraction pattern of Compound 1 Form B-HCl.

FIG. 17 is the DSC curve for a representative sample of Compound 1 FormB-HCl.

FIG. 18 is a curve generated by thermogravimetric analysis of arepresentative sample of Compound 1 Form B-HCl that presents sampleweight as a function of temperature.

FIG. 19 is an FTIR spectrum of a representative sample of Compound 1Form B-HCl.

FIG. 20 is a solid phase ¹³C NMR spectrum of a representative sample ofCompound 1 Form B-HCl.

FIG. 21 is solid phase ¹⁹F NMR spectrum of a representative sample ofCompound 1 Form B-HCl.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “ABC-transporter” as used herein means an ABC-transporterprotein or a fragment thereof comprising at least one binding domain,wherein said protein or fragment thereof is present in vivo or in vitro.The term “binding domain” as used herein means a domain on theABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. etal., J. Gen. Physiol. (1998):111(3), 477-90.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR, R117H CFTR, andG551D CFTR (see, e.g., http://www.genet.sickkids.on.ca/cftr/app, forCFTR mutations).

The term “modulating” as used herein means increasing or decreasing by ameasurable amount.

The term “normal CFTR” or “normal CFTR function” as used herein meanswild-type like CFTR without any impairment due to environmental factorssuch as smoking, pollution, or anything that produces inflammation inthe lungs.

The term “reduced CFTR” or “reduced CFTR function” as used herein meansless than normal CFTR or less than normal CFTR function.

The term “crystalline” refers to compounds or compositions where thestructural units are arranged in fixed geometric patterns or lattices,so that crystalline solids have rigid long range order. The structuralunits that constitute the crystal structure can be atoms, molecules, orions. Crystalline solids show definite melting points.

The term “substantially crystalline” refers to a solid material that ispredominately arranged in fixed geometric patterns or lattices that haverigid long range order. For example, substantially crystalline materialshave more than about 85% crystallinity (e.g., more than about 90%crystallinity, more than about 95% crystallinity, or more than about 99%crystallinity). It is also noted that the term ‘substantiallycrystalline’ includes the descriptor ‘crystalline’, which is defined inthe previous paragraph.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75th Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausalito:1999, and “March'sAdvanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J.,John Wiley & Sons, New York:2001, the entire contents of which arehereby incorporated by reference.

The term “stable”, as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and preferably their recovery, purification, anduse for one or more of the purposes disclosed herein. In someembodiments, a stable compound or chemically feasible compound is onethat is not substantially altered when kept at a temperature of 40° C.or less, in the absence of moisture or other chemically reactiveconditions, for at least a week.

Examples of suitable solvents that may be used in this invention are,but not limited to water, methanol, dichloromethane (DCM), acetonitrile,dimethylformamide (DMF), methyl acetate (MeOAc), ethyl acetate (EtOAc),isopropyl acetate (IPAc), t-butyl acetate (t-BuOAc), isopropyl alcohol(IPA), tetrahydrofuran (THF), methyl ethyl ketone (MEK), t-butanol,diethyl ether (Et₂O), methyl-t-butyl ether (MTBE),1,4-dioxane andN-methylpyrrolidone (NMP).

Examples of suitable coupling agents that may be used in this inventionare, but not limited to 1-(3-(dimethylamino)propyl)-3-ethyl-carbodiimidehydrochloride (EDCI),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), 1-hydroxybenzotriazole (HOBT),2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU), 2-chloro-1,3-dimethyl-2-imidazoliumtetrafluoroborate,1-H-benzotriazolium-1-[bis(dimethylamino)methylene]-5-chlorohexafluorophosphate(HCTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine, and 2-propane phosphonicanhydride (T3P®).

Examples of suitable bases that may be used in this invention are, butnot limited to potassium carbonate (K₂CO₃), N-methylmorpholine (NMM),triethylamine (Et₃N; TEA), diisopropyl-ethyl amine (i-Pr₂EtN; DIEA),pyridine, potassium hydroxide (KOH), sodium hydroxide (NaOH), and sodiummethoxide (NaOMe; NaOCH₃).

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C or ¹⁴C are withinthe scope of this invention. Such compounds are useful, for example, asanalytical tools or probes in biological assays.

Processes of the Invention

In general, the invention provides processes for the synthesis ofcompounds useful as modulators of CFTR.

Preparation of Compound 1

In some embodiments, the invention provides a process for making acrystalline form of Compound 1:

comprising:

(a) reacting Compound 2 with Compound 3 in the presence of a couplingagent

wherein the coupling agent is selected from the group consisting of2-chloro-1,3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU,2-chloro-4,6-dimethoxy-1,3,5-triazine, HATU, HOBT/EDC, and T3P®.

In another aspect of this embodiment, Compound 3 can be the HCl salt.

In one aspect of this embodiment, the coupling of the Compound 2 andCompound 3 is performed in the presence of a base such as K₂CO₃,Et₃N,N-methylmorpholine (NMM), pyridine or diisopropylethyl amine(DIEA).

In another aspect of this embodiment, the coupling of Compound 2 andCompound 3 is performed in the presence of pyridine or DIEA.

In another aspect of this embodiment, the coupling of Compound 2 andCompound 3 is performed in the presence of pyridine.

In another aspect of this embodiment, the coupling of Compound 2 andCompound 3 is performed in the presence of a solvent. In one aspect, thesolvent is a polar aprotic solvent. For example, the solvent is selectedfrom the group consisting of ethyl acetate, isopropyl acetate,tetrahydrofuran, methylethyl ketone, N-Methyl-2-pyrrolidone,acetonitrile, N,N-dimethyl formamide, or 2-methyltetrahydrofuran. Moreparticularly, the solvent is 2-methyltetrahydrofuran.

In another aspect of this embodiment, the coupling of Compound 2 andCompound 3 is performed at a reaction temperature that is maintainedbetween 30° C. and 80° C. In further aspects, the coupling of Compound 2and he aniline of Formula 3 is performed at a reaction temperature thatis maintained between 30° C. and 80° C. (e.g., between about 40° C. and78° C., between about 45° C. and 75° C., between about 50° C. and 70°C., between about 62° C. and 68° C., or about 65° C.) For example, thecoupling of Compound 2 and Compound 3 is performed at a reactiontemperature that is maintained at about 65° C.

In another aspect of this embodiment, Compound 1 is solid Form A.

In another embodiment, the invention provides Compound 1 prepared by theprocess described in the previous paragraphs.

Preparation of Compound 1 Form A

In another embodiment, the invention provides a process for producingCompound 1 having solid Form A,

reacting Compound 2 with Compound 3 in the presence of a coupling agent

wherein the coupling agent is selected from the group consisting of2-chloro-1,3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU,2-chloro-4,6-dimethoxy-1,3,5-triazine, HATU, HOBT/EDC, and T3P®.

In some other aspects of this embodiment, Compound 3 can be the HClsalt.

In one aspect of this embodiment, the coupling of Compound 2 andCompound 3 is performed in the presence of a base such as K₂CO₃,Et₃N,N-methylmorpholine (NMM), pyridine or diisopropylethyl amine(DIEA).

In another aspect of this embodiment, Compound 2 and Compound 3 isperformed in the presence of pyridine or DIEA.

In another aspect of this embodiment, the coupling of Compound 2 andCompound 3 is performed in the presence of pyridine.

In another aspect of this embodiment, coupling of Compound 2 andCompound 3 is performed in the presence of a polar aprotic solvent. Forexample, the polar aprotic solvent is selected from the group consistingof ethyl acetate, isopropyl acetate, tetrahydrofuran, methylethylketone, N-Methyl-2-pyrrolidone, acetonitrile, N,N-dimethyl formamide, or2-methyltetrahydrofuran. More particularly, the coupling of Compound 2and Compound 3 is performed in the presence of a2-methyltetrahydrofuran.

In another aspect of this embodiment, the coupling of Compound 2 andCompound 3 is performed at a reaction temperature that is maintainedbetween 30° C. and 80° C. In further aspects, the coupling of Compound 2and he aniline of Formula 3 is performed at a reaction temperature thatis maintained between 30° C. and 80° C. (e.g., between about 40° C. and78° C., between about 45° C. and 75° C., between about 50° C. and 70°C., between about 62° C. and 68° C., or about 65° C.) For example, thecoupling of Compound 2 and Compound 3 is performed at a reactiontemperature that is maintained at about 65° C.

In another embodiment, the invention provides Compound 1 Form A preparedby the process described in the preceding paragraphs.

In one embodiment, the invention provides a process for preparingCompound 1 Form A, comprising reacting Compound 2 with Compound 3 in thepresence of T3P® and pyridine at a temperature of about 65° C. for about10 hours in the solvent, 2-MeTHF.

Preparation of Compound 1 Form B

In another embodiment, the invention provides a process for producingCompound 1 having solid form Form B:

comprising:

(a) reacting Compound 2 with the hydrochloride salt of Compound 3(3-HCl) in the presence of a coupling agent selected from the groupconsisting of 2-chloro-1,3-dimethyl-2-imidazolium tetrafluoroborate,HBTU, HCTU, 2-chloro-4,6-dimethoxy-1,3,5-triazine, HATU, HOBT/EDC, andT3P®

In one aspect of this embodiment, the coupling of Compound 2 and thehydrochloride salt 3-HCl is performed in the presence of a base such asK₂CO₃, Et₃N,N-methylmorpholine (NMM), pyridine or DIEA.

In some other aspects of this embodiment, the hydrochloride salt 3-HClcan be Compound 3.

In another aspect of this embodiment, the coupling of Compound 2 and thehydrochloride salt 3-HCl is performed in the presence of pyridine orDIEA.

In another aspect of this embodiment, the coupling of Compound 2 and thehydrochloride salt 3-HCl is performed in the presence of pyridine.

In another aspect of this embodiment, the coupling of Compound 2 and thehydrochloride salt 3-HCl is performed in the presence of a solvent suchas EtOAc, IPAc, THF, MEK, NMP, acetonitrile, DMF, or2-methyltetrahydrofuran. More particularly, the coupling of Compound 2and the hydrochloride salt 3-HCl is performed in the presence of a2-methyltetrahydrofuran.

In another aspect of this embodiment, the coupling of Compound 2 and thehydrochloride salt 3-HCl is s performed at a reaction temperature thatis maintained between 15° C. and 70° C. In further aspects, the couplingof Compound 2 and the hydrochloride salt 3-HCl is performed at areaction temperature that is maintained between 15° C. and 70° C. (e.g.,between about 20° C. and 65° C., between about 25° C. and 60° C.,between about 30° C. and 55° C., between about 35° C. and 50° C., orabout 45° C.). In a preferred aspect of this embodiment, the coupling ofCompound 2 and the HCl salt of Compound 3 is performed at a reactiontemperature that is maintained at about 45° C.

In another aspect, the coupling reaction is stirred for a duration offrom about 1.5 hours to about 10 hours. In further aspects, the couplingreaction is stirred for a duration of from about 1.5 hours to about 10hours (e.g., from about 2 hours to about 7 hours, from about 3 hours toabout 6 hours, from about 4 hours to about 5.5 hours, or about 5 hours).

In another embodiment, the invention provides Compound 1 Form B preparedby the process of described in the preceding paragraphs.

In one embodiment, the invention provides a process for preparingCompound 1 Form B, comprising reacting Compound 2 with Compound 3-HCl inthe presence of T3P® and pyridine at a temperature of about 45° C. forabout 5-6 hours in the solvent, 2-MeTHF.

Preparation of Compound 1 Form A-HCl

In another embodiment, the invention provides a process for producingthe hydrochloride salt of Compound 1 having solid Form A-HCl:

comprising:

(a) reacting Compound 2 with the hydrochloride salt of Compound 3(3-HCl) in the presence of a coupling agent selected from the groupconsisting of 2-chloro-1,3-dimethyl-2-imidazolium tetrafluoroborate,HBTU, HCTU, 2-chloro-4,6-dimethoxy-1,3,5-triazine, HATU, HOBT/EDC, andT3P®:

and

(b) treating a mixtures of the product of step (a) with HCl.

In one aspect of this embodiment, step (a) is performed in the presenceof a base such as K₂CO₃, Et₃N,N-methylmorpholine (NMM), pyridine ordiisopropylethyl amine (DIEA).

In another aspect of this embodiment, step (a) is performed in thepresence of pyridine or DIEA.

In another aspect of this embodiment, step (a) is performed in thepresence of pyridine.

In another aspect of this embodiment, step (a) is performed in thepresence of a polar aprotic solvent. For example, the polar aproticsolvent is selected from the group consisting of ethyl acetate,isopropyl acetate, tetrahydrofuran, methylethyl ketone,N-Methyl-2-pyrrolidone, acetonitrile, N,N-dimethyl formamide, or2-methyltetrahydrofuran. More particularly, the coupling of Compound 2and 3-HCl is performed in the presence of a 2-methyltetrahydrofuran.

In another aspect of this embodiment, step (a) is performed at areaction temperature that is maintained between 10° C. and 80° C. Inanother aspect of this embodiment, step (a) is performed at a reactiontemperature that is maintained between 15° C. and 70° C. In furtheraspects, the coupling of Compound 2 and the HCl salt of Compound 3 isperformed at a reaction temperature that is maintained between 15° C.and 70° C. (e.g., between about 20° C. and 65° C., between about 25° C.and 60° C., between about 30° C. and 55° C., between about 35° C. and50° C., or about 45° C.) For example, the coupling of Compound 2 and3-HCl is performed at a reaction temperature that is maintained at about45° C.

In another aspect of this embodiment, the time of step (a) is from about1.5 hours to about 72 hours. In further aspects, the coupling reactionis stirred for a duration of from about 1.5 hours to about 72 hours ormore (e.g., from about 2 hours to about 48 hours, from about 3 hours toabout 24 hours, from about 5 hours to about 20 hours, or from about 12hours to about 15 hours).

In a further aspect of this embodiment, the coupling product in step (a)is treated with hydrogen chloride (HCl) in step (b). For example, HClgas is bubbled into a mixture comprising the product of the couplingreaction of step (a) and apolar aprotic solvent such as2-methyltetrahydrofuran.

Typically at least about 1 equivalent of HCl gas, and up to about 50equivalents of HCl gas is bubbled into the mixture. More typically, fromabout 2 equivalents (eq.) to about 20 equivalents (from about of 5 eq toabout 15 eq, from about 8 eq to about 12 eq, or about 10 eq) of HCl gasis bubbled into a reaction mixture comprising the coupling product ofstep (a).

Typically, the HCl gas is bubbled into the mixture product of step (a)and a solvent, such as an aprotic solvent, for a period of from about0.5 hours to about 5 hours, and more typically, for a period of fromabout 0.5 hours to about 5 hours (e.g., from about 0.75 hours to about 3hours, or about 2 hours).

In another embodiment, the invention provides Compound 1 Form A-HClprepared by the process described in the preceding paragraphs.

In one embodiment, the invention provides a process for preparingCompound 1 Form A-HCl, comprising reacting Compound 2 with Compound3-HCl in the presence of T3P® and pyridine at a temperature of about 45°C. for about 12-15 hours in the solvent, 2-MeTHF, followed by treatmentwith gaseous HCl.

Preparation of Compound 1 Form B-HCl

In another aspect, the invention provides a process for producing ahydrochloride salt of Compound 1, having the solid form Form B-HCl:

comprising:

(a) mixing a hydrochloride salt of Compound 1 Form A-HCl, as describedherein, with an organic solvent and water to generate a mixture:

and

(b) heating the mixture.

In one aspect of this embodiment, the organic solvent comprises dimethylsulfoxide, dimethylformamide, dioxane, hexamethylphosphorotriamide,tetrahydrofuran, EtOAc, IPAc, THF, MEK, NMP, acetonitrile, DMF, EtOH,MeOH, isopropyl alcohol, or 2-methyltetrahydrofuran. More particularly,the aprotic solvent comprises 2-methyltetrahydrofuran.

In another aspect, the mixing in step (a) is followed in step (b) withmaintaining the mixture at a temperature of from about 30° C. to about80° C. (e.g., from about 40° C. to about 70° C., from about 50° C. toabout 65° C., or about 60° C.) The process of claim 40-43, wherein themixture is maintained at a temperature of 30° C. to about 80° C. for aperiod of from about 12 hours to about 72 hours.

In another aspect, after heating in step (b), the mixture is filtered togenerate a filter cake.

In another aspect, the filtering is followed by washing the filter cakewith an aprotic solvent such as 2-methyltetrahydrofuran.

In another embodiment, the invention provides Compound 1 Form B-HClprepared by the process described in the preceding paragraphs.

In one embodiment, the invention provides a process for preparingCompound 1 Form B-HCl, comprising heating Compound 1 Form A-HCl in amixture of 2-MeTHF and water at a temperature of 60° C. for 48 hours;cooling to room temperature and filtering precipitated product; anddrying the produce under vacuum at a temperature of 60° C.

In another embodiment, the invention provides a process for preparingCompound 1 Form B-HCl, comprising heating Compound 1 Form A-HCl in amixture of EtOH and water to reflux temperature; cooling to 20° C. andstirring for 3 hours; filtering precipitated product; and drying theproduce under vacuum at a temperature of 45° C.

Preparation of Compounds 2, 3, and 3-HCl

In another embodiment, the invention provides a method for preparingCompound 2:

comprising:

(a) reacting Compound 4 with diethyl 2-(ethoxymethylene)malonate 5 togenerate ester 6A

and

(b) treating ester 6A with a source of atomic hydrogen, such as hydrogengas or formate in the presence of a catalyst and a base in separatesteps to generate Compound 2

In one aspect, Compound 6A is treated with a source of atomic hydrogen,such as hydrogen gas or formate in the presence of a catalyst to produceCompound 6C

which is then treated with a base to produce Compound 2.

In another aspect, Compound 6A is treated with a base to produceCompound 6D

which is then treated with a source of atomic hydrogen, such as hydrogengas or formate in the presence of a catalyst to produce Compound 2.

In some aspects, the source of hydrogen is hydrogen gas. In otheraspects, the source of hydrogen is formate.

In some aspects, the catalyst is a palladium catalyst.

In some aspects, the base is aqueous sodium hydroxide or aqueouspotassium hydroxide. For example, the base is aqueous sodium hydroxide.

In one embodiment, the invention provides a process for preparingCompound 2 comprising the steps of:

-   -   a) reacting Compound 4 with Compound 5 in toluene at reflux        temperature in a Dean-Stark apparatus to form Compound 6B;    -   b) heating Compound 6B in Dowtherm, at a temperature of about        260° C. to produce the cyclized product, Compound 6A;    -   c) hydrogenating Compound 6A in EtOH using Pd/C as a catalyst in        the presence of hydrogen gas and triethylamine to produce        Compound 6C; and    -   d) hydrolyzing Compound 6C using 5 M NaOH to produce Compound 2

In one embodiment, the invention provides a process for preparingCompound 2 comprising the steps of:

-   -   a) reacting Compound 4 with Compound 5 in toluene at reflux        temperature in a Dean-Stark apparatus to form Compound 6B;    -   b) heating Compound 6B in Dowtherm, at a temperature of about        260° C. to produce the cyclized product, Compound 6A;    -   c) hydrolyzing Compound 6A using NaOH in a mixture of        isopropanol and water to produce Compound 6D; and    -   d) hydrogenating Compound 6D in EtOH using Pd/C as a catalyst in        the presence of formate, sodium methoxide and methanol; and    -   e) acidifying the solution with acetic acid to produce Compound        2

In another embodiment, the invention provides a method for preparingCompound 3

comprising:

(a) reacting compound 7, wherein Hal is F, Cl, Br or I, with7-azabicyclo[2.2.1]heptane 8, or a salt thereof, to generate compound 9

and

(b) reducing the nitro moiety in compound 9 to provide aniline 3.

In one aspect of this embodiment, step (a) is performed in the presenceof a base in a polar aprotic solvent. For example, the base is atertiary amine bases such as triethyl amine, or diisopropylethyl amineor the like and the solvent such as acetonitrile.

In another aspect of this embodiment, step (b) is performed usinghydrogen gas in the and a transition metal catalyst in an alcoholicsolvent. For example, the catalyst comprises a group 9 or group 10transition metal catalyst derived from Pt, Pd, or Ni. More particularly,the catalyst comprises Pd. The alcoholic solvent comprises an alcoholsuch as isopropanol, ethanol, methanol, or the like. For example, thesolvent comprises ethanol.

In another embodiment, the invention provides a method for preparingCompound 3:

comprising:

(a) reacting Compound 7, wherein Hal is F, Cl, Br or I, with thehydrochloride salt of 7-azabicyclo[2.2.1]heptane (8-HCl) to generatecompound 9

and

(b) reducing the nitro moiety in Compound 9 to provide Compound 3.

In one aspect of this embodiment, step (a) is performed in the presenceof an inorganic carbonate base such as sodium carbonate and a polaraprotic solvent such as DMSO.

In another aspect of this embodiment, step (b) is performed usinghydrogen gas in the and a transition metal catalyst in an alcoholicsolvent. For example, the catalyst comprises a group 9 or group 10transition metal catalyst derived from Pt, Pd, or Ni. More particularly,the catalyst comprises Pd. The alcoholic solvent comprises an alcoholsuch as isopropanol, ethanol, methanol, or the like. For example, thesolvent comprises ethanol.

In another embodiment, the invention provides a method for preparing thehydrochloride salt 3-HCl:

comprising:

(a) reacting compound 7, wherein Hal is F, Cl, Br or I, with7-azabicyclo[2.2.1]heptane hydrochloride salt 8-HCl to generate compound9

(b) reducing the nitro moiety in compound 9 to provide aniline 3;

and

(c) treating the product of step (c) with HCl gas to provide 3-HCl.

In one aspect of this embodiment, step (a) is performed in the presenceof an inorganic carbonate base such as sodium carbonate and a polaraprotic solvent such as DMSO.

In another aspect of this embodiment, step (b) is performed usinghydrogen gas in the and a transition metal catalyst in an alcoholicsolvent. For example, the catalyst comprises a group 9 or group 10transition metal catalyst derived from Pt, Pd, or Ni. More particularly,the catalyst comprises Pd. The alcoholic solvent comprises an alcoholsuch as isopropanol, ethanol, methanol, or the like. For example, thesolvent comprises ethanol.

In another embodiment, the invention includes a method for preparingCompound 8,

-   -   or a pharmaceutically acceptable salt thereof, comprising        contacting trans-4-aminocyclohexanol with Boc anhydride to        produce a compound of formula A

-   -   contacting a compound of formula A with methanesulfonic acid to        produce a compound of formula B

-   -   contacting a compound of formula B with trifluoroacetic acid to        produce a compound of formula C

-   -    and    -   contacting a compound of formula C with hydroxide to produce a        compound of formula 8.

In another embodiment, the invention includes a method of producing acompound of formula 8-HCl,

comprising contacting a compound of formula 8 with hydrochloric acid.

In one embodiment, the invention provides a process for producingCompound 3, comprising the steps of:

-   -   a) reacting Compound 7 with Compound 8-HCl in acetonitrile, in        the presence of triethylamine at about 80° C. for about 16 hours        to produce Compound 9; and    -   b) hydrogenating Compound 9 in Ethanol, using Pd/C as a        catalysts in the presence of hydrogen gas

In one embodiment, the invention provides a process for producingCompound 3, comprising the steps of:

-   -   a) reacting Compound 7 with Compound 8-HCl in DMSO, in the        presence of sodium carbonate at about 55° C. to produce Compound        9; and    -   b) hydrogenating Compound 9 in Ethanol, using Pd/C as a        catalysts in the presence of hydrogen gas

In one embodiment, the invention provides a process for producingCompound 3, comprising the steps of:

-   -   a) reacting Compound 7 with Compound 8-HCl in dichloromethane,        in the presence of sodium hydroxide and tetrabutylammonium        bromide to produce Compound 9; and    -   b) hydrogenating Compound 9 in Ethanol, using Pd/C as a        catalysts in the presence of hydrogen gas

In one embodiment, the invention provides a process for producingCompound 3-HCl, comprising hydrogenating the hydrochloride salt ofCompound 9 in 2-MeTHF in the presence of hydrogen gas using Pd/C as acatalyst.

Other Aspects of the Present Invention

In one aspect, the invention features a pharmaceutical compositioncomprising Compound 1 Form A, Compound 1 Form A-HCl, Compound 1 Form B,Compound 1 Form B-HCl, or any combination thereof, and apharmaceutically acceptable adjuvant or carrier.

In one aspect, the present invention features a method of treating aCFTR mediated disease in a human comprising administering to the humanan effective amount of Compound 1 Form A, Compound 1 Form A-HCl,Compound 1 Form B, Compound 1 Form B-HCl, or any combination thereof.

In some embodiments, the method comprises administering an additionaltherapeutic agent.

In certain embodiments, the present invention provides a method oftreating diseases associated with reduced CFTR function due to mutationsin the gene encoding CFTR or environmental factors (e.g., smoke). Thesediseases include, cystic fibrosis, asthma, smoke induced COPD, chronicbronchitis, rhinosinusitis, constipation, pancreatitis, pancreaticinsufficiency, male infertility caused by congenital bilateral absenceof the vas deferens (CBAVD), mild pulmonary disease, idiopathicpancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liverdisease, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington's, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease, Osteoporosis, Osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, and PrimaryCiliary Dyskinesia (PCD), a term for inherited disorders of thestructure and/or function of cilia, including PCD with situs inversus(also known as Kartagener syndrome), PCD without situs inversus andciliary aplasia.

In some embodiments, the method includes treating or lessening theseverity of cystic fibrosis in a patient comprising administering tosaid patient one of the compositions as defined herein. In certainembodiments, the patient possesses mutant forms of human CFTR. In otherembodiments, the patient possesses one or more of the followingmutations ΔF508, R117H, and G551D of human CFTR. In one embodiment, themethod includes treating or lessening the severity of cystic fibrosis ina patient possessing the ΔF508 mutation of human CFTR comprisingadministering to said patient one of the compositions as defined herein.In one embodiment, the method includes treating or lessening theseverity of cystic fibrosis in a patient possessing the G551D mutationof human CFTR comprising administering to said patient one of thecompositions as defined herein. In one embodiment, the method includestreating or lessening the severity of cystic fibrosis in a patientpossessing the ΔF508 mutation of human CFTR on at least one allelecomprising administering to said patient one of the compositions asdefined herein. In one embodiment, the method includes treating orlessening the severity of cystic fibrosis in a patient possessing theΔF508 mutation of human CFTR on both alleles comprising administering tosaid patient one of the compositions as defined herein. In oneembodiment, the method includes treating or lessening the severity ofcystic fibrosis in a patient possessing the G551D mutation of human CFTRon at least one allele comprising administering to said patient one ofthe compositions as defined herein. In one embodiment, the methodincludes treating or lessening the severity of cystic fibrosis in apatient possessing the G551D mutation of human CFTR on both allelescomprising administering to said patient one of the compositions asdefined herein.

In some embodiments, the method includes lessening the severity ofcystic fibrosis in a patient comprising administering to said patientone of the compositions as defined herein. In certain embodiments, thepatient possesses mutant forms of human CFTR. In other embodiments, thepatient possesses one or more of the following mutations ΔF508, R117H,and G551D of human CFTR. In one embodiment, the method includeslessening the severity of cystic fibrosis in a patient possessing theΔF508 mutation of human CFTR comprising administering to said patientone of the compositions as defined herein. In one embodiment, the methodincludes lessening the severity of cystic fibrosis in a patientpossessing the G551D mutation of human CFTR comprising administering tosaid patient one of the compositions as defined herein. In oneembodiment, the method includes lessening the severity of cysticfibrosis in a patient possessing the ΔF508 mutation of human CFTR on atleast one allele comprising administering to said patient one of thecompositions as defined herein. In one embodiment, the method includeslessening the severity of cystic fibrosis in a patient possessing theΔF508 mutation of human CFTR on both alleles comprising administering tosaid patient one of the compositions as defined herein. In oneembodiment, the method includes lessening the severity of cysticfibrosis in a patient possessing the G551D mutation of human CFTR on atleast one allele comprising administering to said patient one of thecompositions as defined herein. In one embodiment, the method includeslessening the severity of cystic fibrosis in a patient possessing theG551D mutation of human CFTR on both alleles comprising administering tosaid patient one of the compositions as defined herein.

In some aspects, the invention provides a method of treating orlessening the severity of Osteoporosis in a patient comprisingadministering to said patient Compound 1 as described herein.

In certain embodiments, the method of treating or lessening the severityof Osteoporosis in a patient comprises administering to said patient apharmaceutical composition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of Osteopenia in a patient comprisingadministering to said patient Compound 1 as described herein.

In certain embodiments, the method of treating or lessening the severityof Osteopenia in a patient comprises administering to said patient apharmaceutical composition as described herein.

In some aspects, the invention provides a method of bone healing and/orbone repair in a patient comprising administering to said patientCompound 1 as described herein.

In certain embodiments, the method of bone healing and/or bone repair ina patient comprises administering to said patient a pharmaceuticalcomposition as described herein.

In some aspects, the invention provides a method of reducing boneresorption in a patient comprising administering to said patientCompound 1 as described herein.

In certain embodiments, the method of reducing bone resorption in apatient comprises administering to said patient a pharmaceuticalcomposition as described herein.

In some aspects, the invention provides a method of increasing bonedeposition in a patient comprising administering to said patientCompound 1 as described herein.

In certain embodiments, the method of increasing bone deposition in apatient comprises administering to said patient a pharmaceuticalcomposition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of COPD in a patient comprising administering tosaid patient Compound 1 as described herein.

In certain embodiments, the method of treating or lessening the severityof COPD in a patient comprises administering to said patient apharmaceutical composition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of smoke induced COPD in a patient comprisingadministering to said patient Compound 1 as described herein.

In certain embodiments, the method of treating or lessening the severityof smoke induced COPD in a patient comprises administering to saidpatient a pharmaceutical composition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of chronic bronchitis in a patient comprisingadministering to said patient Compound 1 as described herein.

In certain embodiments, the method of treating or lessening the severityof chronic bronchitis in a patient comprises administering to saidpatient a pharmaceutical composition as described herein.

In one embodiment, the present invention provides a method of treatingcystic fibrosis in a human, comprising administering to said human aneffective amount of Compound 1 Form A, Form A-HCl, Form B, Form B-HCl,or any combination thereof.

In one aspect, the present invention features a pharmaceutical pack orkit comprising Compound 1 Form A, Form A-HCl, Form B, Form B-HCl, or anycombination of these forms, and a pharmaceutically acceptable carrier.

In one aspect, the invention features a crystal form ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamidehaving a trigonal crystal system, a R-3 space group, and the followingunit cell dimensions: a=19.1670(4) Å, b=19.1670(4) Å, c=33.6572(12) Å,α=90°, β=90°, and γ=120°.

In one embodiment, the present invention provides a crystal ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamidein Form B having a monoclinic crystal system, a P21/c space group, andthe following unit cell dimensions: a=13.5429(4) Å, b=13.4557(4) Å,c=12.0592(4) Å, α=90°, β=101.193°, and γ=90°.

Uses, Formulation and Administration

In one aspect of the present invention, pharmaceutically acceptablecompositions are provided, wherein these compositions comprise Form A asdescribed herein, and optionally comprise a pharmaceutically acceptablecarrier, adjuvant or vehicle. In certain embodiments, these compositionsoptionally further comprise one or more additional therapeutic agents.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating or lessening the severity of a condition, disease, or disorderimplicated by CFTR mutation. In certain embodiments, the presentinvention provides a method of treating a condition, disease, ordisorder implicated by a deficiency of the CFTR activity, the methodcomprising administering composition comprising Compound 1 Form A, FormA-HCl, Form B, Form B-HCl, or any combination of these forms, to asubject, preferably a mammal, in need thereof.

In certain embodiments, the present invention provides a method oftreating diseases associated with reduced CFTR function due to mutationsin the gene encoding CFTR or environmental factors (e.g., smoke). Thesediseases include, cystic fibrosis, chronic bronchitis, recurrentbronchitis, acute bronchitis, male infertility caused by congenitalbilateral absence of the vas deferens (CBAVD), female infertility causedby congenital absence of the uterus and vagina (CAUV), idiopathicchronic pancreatitis (ICP), idiopathic recurrent pancreatitis,idiopathic acute pancreatitis, chronic rhinosinusitis, primarysclerosing cholangitis, allergic bronchopulmonary aspergillosis,diabetes, dry eye, constipation, allergic bronchopulmonary aspergillosis(ABPA), bone diseases (e.g., osteoporosis), and asthma.

In certain embodiments, the present invention provides a method fortreating diseases associated with normal CFTR function. These diseasesinclude, chronic obstructive pulmonary disease (COPD), chronicbronchitis, recurrent bronchitis, acute bronchitis, rhinosinusitis,constipation, pancreatitis including chronic pancreatitis, recurrentpancreatitis, and acute pancreatitis, pancreatic insufficiency, maleinfertility caused by congenital bilateral absence of the vas deferens(CBAVD), mild pulmonary disease, idiopathic pancreatitis, liver disease,hereditary emphysema, gallstones, gasgtro-esophageal reflux disease,gastrointestinal malignancies, inflammatory bowel disease, constipation,diabetes, arthritis, osteoporosis, and osteopenia.

In certain embodiments, the present invention provides a method fortreating diseases associated with normal CFTR function includinghereditary hemochromatosis, coagulation-fibrinolysis deficiencies, suchas protein C deficiency, Type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, Type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetesinsipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Toothsyndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases suchas Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis, progressive supranuclear palsy, Pick's disease, severalpolyglutamine neurological disorders such as Huntington's,spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, Gorham's Syndrome, chloridechannelopathies, myotonia congenita (Thomson and Becker forms),Bartter's syndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, PrimaryCiliary Dyskinesia (PCD), PCD with situs inversus (also known asKartagener syndrome), PCD without situs inversus and ciliary aplasia, orSjogren's disease, comprising the step of administering to said mammalan effective amount of a composition comprising Compound 1 Form A,Compound 1 Form A-HCl, Compound 1 Form B, Compound 1 Form B-HCl, or anycombination of these forms, described herein.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis comprising the step ofadministering to said mammal a composition comprising the step ofadministering to said mammal an effective amount of a compositioncomprising Compound 1 Form A, Compound 1 Form A-HCl, Compound 1 Form B,Compound 1 Form B-HCl, or any combination of these forms, describedherein.

According to the invention an “effective amount” of Compound 1 Form A,Compound 1 Form A-HCl, Compound 1 Form B, Compound 1 Form B-HCl, anycombination of these forms, or a pharmaceutically acceptable compositionthereof is that amount effective for treating or lessening the severityof one or more of the diseases, disorders or conditions as recitedabove.

Compound 1 Form A, Compound 1 Form A-HCl, Compound 1 Form B, Compound 1Form B-HCl, or any combination of these forms, or a pharmaceuticallyacceptable composition thereof may be administered using any amount andany route of administration effective for treating or lessening theseverity of one or more of the diseases, disorders or conditions asrecited above.

In certain embodiments, Compound 1 Form A, Compound 1 Form A-HCl,Compound 1 Form B, Compound 1 Form B-HCl, any combination of theseforms, or a pharmaceutically acceptable composition thereof is usefulfor treating or lessening the severity of cystic fibrosis in patientswho exhibit residual CFTR activity in the apical membrane of respiratoryand non-respiratory epithelia. The presence of residual CFTR activity atthe epithelial surface can be readily detected using methods known inthe art, e.g., standard electrophysiological, biochemical, orhistochemical techniques. Such methods identify CFTR activity using invivo or ex vivo electrophysiological techniques, measurement of sweat orsalivary Cl³¹ concentrations, or ex vivo biochemical or histochemicaltechniques to monitor cell surface density. Using such methods, residualCFTR activity can be readily detected in patients heterozygous orhomozygous for a variety of different mutations, including patientshomozygous or heterozygous for the most common mutation, ΔF508.

In another embodiment, Compound 1 Form A, Compound 1 Form A-HCl,Compound 1 Form B, Compound 1 Form B-HCl, or any combination of theseforms, described herein or a pharmaceutically acceptable compositionthereof is useful for treating or lessening the severity of cysticfibrosis in patients who have residual CFTR activity induced oraugmented using pharmacological methods or gene therapy. Such methodsincrease the amount of CFTR present at the cell surface, therebyinducing a hitherto absent CFTR activity in a patient or augmenting theexisting level of residual CFTR activity in a patient.

In one embodiment, Compound 1 Form A, Compound 1 Form A-HCl, Compound 1Form B, Compound 1 Form B-HCl, or any combination of these forms,described herein, or a pharmaceutically acceptable composition thereofis useful for treating or lessening the severity of cystic fibrosis inpatients within certain genotypes exhibiting residual CFTR activity,e.g., class III mutations (impaired regulation or gating), class IVmutations (altered conductance), or class V mutations (reducedsynthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II, III, IV,and V cystic fibrosis Tansmembrane Conductance Regulator Defects andOpportunities of Therapy; Current Opinion in Pulmonary Medicine6:521-529, 2000). Other patient genotypes that exhibit residual CFTRactivity include patients homozygous for one of these classes orheterozygous with any other class of mutations, including class Imutations, class II mutations, or a mutation that lacks classification.

In one embodiment, Compound 1 Form A, Compound 1 Form A-HCl, Compound 1Form B, Compound 1 Form B-HCl, or any combination of these formsdescribed herein or a pharmaceutically acceptable composition thereof isuseful for treating or lessening the severity of cystic fibrosis inpatients within certain clinical phenotypes, e.g., a moderate to mildclinical phenotype that typically correlates with the amount of residualCFTR activity in the apical membrane of epithelia. Such phenotypesinclude patients exhibiting pancreatic insufficiency or patientsdiagnosed with idiopathic pancreatitis and congenital bilateral absenceof the vas deferens, or mild lung disease.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or patch), bucally, as an oral or nasal spray,or the like, depending on the severity of the infection being treated.In certain embodiments, the compounds of the invention may beadministered orally or parenterally at dosage levels of about 0.01 mg/kgto about 50 mg/kg and preferably from about 0.5 mg/kg to about 25 mg/kg,of subject body weight per day, one or more times a day, to obtain thedesired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are prepared by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

It will also be appreciated that the Form A, Form A-HCl, Form B, FormB-HCl, or any combination thereof described herein or a pharmaceuticallyacceptable composition thereof can be employed in combination therapies,that is, Form A, Form A-HCl, Form B, Form B-HCl, or any combinationthereof described herein or a pharmaceutically acceptable compositionthereof can be administered concurrently with, prior to, or subsequentto, one or more other desired therapeutics or medical procedures. Theparticular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, an inventive compound may be administered concurrently withanother agent used to treat the same disorder), or they may achievedifferent effects (e.g., control of any adverse effects). As usedherein, additional therapeutic agents that are normally administered totreat or prevent a particular disease, or condition, are known as“appropriate for the disease, or condition, being treated.”

In one embodiment, the additional agent is selected from a mucolyticagent, bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than a compound of thepresent invention, or a nutritional agent.

In one embodiment, the additional agent is an antibiotic. Exemplaryantibiotics useful herein include tobramycin, including tobramycininhaled powder (TIP), azithromycin, aztreonam, including the aerosolizedform of aztreonam, amikacin, including liposomal formulations thereof,ciprofloxacin, including formulations thereof suitable foradministration by inhalation, levoflaxacin, including aerosolizedformulations thereof, and combinations of two antibiotics, e.g.,fosfomycin and tobramycin.

In another embodiment, the additional agent is a mucolyte. Exemplarymucolytes useful herein includes Pulmozyme®.

In another embodiment, the additional agent is a bronchodialator.Exemplary bronchodialtors include albuterol, metaprotenerol sulfate,pirbuterol acetate, salmeterol, or tetrabuline sulfate.

In another embodiment, the additional agent is effective in restoringlung airway surface liquid. Such agents improve the movement of salt inand out of cells, allowing mucus in the lung airway to be more hydratedand, therefore, cleared more easily. Exemplary such agents includehypertonic saline, denufosol tetrasodium([[(3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogenphosphate), or bronchitol (inhaled formulation of mannitol).

In another embodiment, the additional agent is an anti-inflammatoryagent, i.e., an agent that can reduce the inflammation in the lungs.Exemplary such agents useful herein include ibuprofen, docosahexanoicacid (DHA), sildenafil, inhaled glutathione, pioglitazone,hydroxychloroquine, or simavastatin.

In another embodiment, the additional agent reduces the activity of theepithelial sodium channel blocker (ENaC) either directly by blocking thechannel or indirectly by modulation of proteases that lead to anincrease in ENaC activity (e.g., seine proteases, channel-activatingproteases). Exemplary such agents include camostat (a trypsin-likeprotease inhibitor), QAU145, 552-02, GS-9411, INO-4995, Aerolytic, andamiloride. Additional agents that reduce the activity of the epithelialsodium channel blocker (ENaC) can be found, for example, in PCTPublication No. WO2009/074575, the entire contents of which areincorporated herein in their entirety.

Amongst other diseases described herein, combinations of CFTRmodulators, such as those described herein, and agents that reduce theactivity of ENaC are use for treating Liddle's syndrome, an inflammatoryor allergic condition including cystic fibrosis, primary ciliarydyskinesia, chronic bronchitis, chronic obstructive pulmonary disease,asthma, respiratory tract infections, lung carcinoma, xerostomia andkeratoconjunctivitis sire, respiratory tract infections (acute andchronic; viral and bacterial) and lung carcinoma.

Combinations of CFTR modulators, such as those described herein, andagents that reduce the activity of ENaC are also useful for treatingdiseases mediated by blockade of the epithelial sodium channel alsoinclude diseases other than respiratory diseases that are associatedwith abnormal fluid regulation across an epithelium, perhaps involvingabnormal physiology of the protective surface liquids on their surface,e.g., xerostomia (dry mouth) or keratoconjunctivitis sire (dry eye).Furthermore, blockade of the epithelial sodium channel in the kidneycould be used to promote diuresis and thereby induce a hypotensiveeffect.

Asthma includes both intrinsic (non-allergic) asthma and extrinsic(allergic) asthma, mild asthma, moderate asthma, severe asthma,bronchitic asthma, exercise-induced asthma, occupational asthma andasthma induced following bacterial infection. Treatment of asthma isalso to be understood as embracing treatment of subjects, e.g., of lessthan 4 or 5 years of age, exhibiting wheezing symptoms and diagnosed ordiagnosable as “wheezy infants”, an established patient category ofmajor medical concern and now often identified as incipient orearly-phase asthmatics. (For convenience this particular asthmaticcondition is referred to as “wheezy-infant syndrome”.) Prophylacticefficacy in the treatment of asthma will be evidenced by reducedfrequency or severity of symptomatic attack, e.g., of acute asthmatic orbronchoconstrictor attack, improvement in lung function or improvedairways hyperreactivity. It may further be evidenced by reducedrequirement for other, symptomatic therapy, i.e., therapy for orintended to restrict or abort symptomatic attack when it occurs, e.g.,anti-inflammatory (e.g., cortico-steroid) or bronchodilatory.Prophylactic benefit in asthma may, in particular, be apparent insubjects prone to “morning dipping”. “Morning dipping” is a recognizedasthmatic syndrome, common to a substantial percentage of asthmatics andcharacterized by asthma attack, e.g., between the hours of about 4-6 am,i.e., at a time normally substantially distant from any previouslyadministered symptomatic asthma therapy.

Chronic obstructive pulmonary disease includes chronic bronchitis ordyspnea associated therewith, emphysema, as well as exacerbation ofairways hyperreactivity consequent to other drug therapy, in particular,other inhaled drug therapy. In some embodiments, the combinations ofCFTR modulators, such as those described herein, and agents that reducethe activity of ENaC are useful for the treatment of bronchitis ofwhatever type or genesis including, e.g., acute, arachidic, catarrhal,croupus, chronic or phthinoid bronchitis.

In another embodiment, the additional agent is a CFTR modulator otherthan Form A, Form B, Form B-HCl, and Form A-HCl, i.e., an agent that hasthe effect of modulating CFTR activity. Exemplary such agents includeataluren (“PTC124®”; 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoicacid), sinapultide, lancovutide, depelestat (a human recombinantneutrophil elastase inhibitor), cobiprostone(7-{(2R,4aR,5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta[b]pyran-5-yl}heptanoicacid), or(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid. In another embodiment, the additional agent is(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.

In another embodiment, the additional agent is a nutritional agent.Exemplary such agents include pancrelipase (pancreating enzymereplacement), including Pancrease®, Pancreacarb®, Ultrase®, or Creon®,Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione inhalation.In one embodiment, the additional nutritional agent is pancrelipase.

In one embodiment, the additional agent is a CFTR modulator other than acompound of the present invention.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

Compound 1 Form A, Form A-HCl, Form B, Form B-HCl, or any combinationthereof described herein or a pharmaceutically acceptable compositionthereof may also be incorporated into compositions for coating animplantable medical device, such as prostheses, artificial valves,vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present invention includes an implantable device coated witha composition comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

Another aspect of the invention relates to modulating CFTR activity in abiological sample or a patient (e.g., in vitro or in vivo), which methodcomprises administering to the patient, or contacting said biologicalsample with Compound 20 Form A, Form A-HCl, Form B, Form B-HCl, or anycombination thereof described herein or a pharmaceutically acceptablecomposition thereof. The term “biological sample”, as used herein,includes, without limitation, cell cultures or extracts thereof;biopsied material obtained from a mammal or extracts thereof; and blood,saliva, urine, feces, semen, tears, or other body fluids or extractsthereof.

Modulation of CFTR in a biological sample is useful for a variety ofpurposes that are known to one of skill in the art. Examples of suchpurposes include, but are not limited to, the study of CFTR inbiological and pathological phenomena; and the comparative evaluation ofnew modulators of CFTR.

In yet another embodiment, a method of modulating activity of an anionchannel in vitro or in vivo, is provided comprising the step ofcontacting said channel with Compound 20 Form A, Form A-HCl, Form B,Form B-HCl, or any combination thereof described herein or apharmaceutically acceptable composition thereof. In preferredembodiments, the anion channel is a chloride channel or a bicarbonatechannel. In other preferred embodiments, the anion channel is a chloridechannel.

According to an alternative embodiment, the present invention provides amethod of increasing the number of functional CFTR in a membrane of acell, comprising the step of contacting said cell with Compound 20 FormA, Form A-HCl, Form B, Form B-HCl, or any combination thereof describedherein or a pharmaceutically acceptable composition thereof.

According to another preferred embodiment, the activity of the CFTR ismeasured by measuring the transmembrane voltage potential. Means formeasuring the voltage potential across a membrane in the biologicalsample may employ any of the known methods in the art, such as opticalmembrane potential assay or other electrophysiological methods.

The optical membrane potential assay utilizes voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells.” Biophys J 69(4):1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997); “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4):269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9):431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission can be monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

In another aspect the present invention provides a kit for use inmeasuring the activity of CFTR or a fragment thereof in a biologicalsample in vitro or in vivo comprising (i) a composition comprisingCompound 20 Form A, Form A-HCl, Form B, Form B-HCl, or any combinationthereof or any of the above embodiments; and (ii) instructions for a)contacting the composition with the biological sample and b) measuringactivity of said CFTR or a fragment thereof. In one embodiment, the kitfurther comprises instructions for a) contacting an additionalcomposition with the biological sample; b) measuring the activity ofsaid CFTR or a fragment thereof in the presence of said additionalcompound, and c) comparing the activity of the CFTR in the presence ofthe additional compound with the density of the CFTR in the presence ofForm A, Form A-HCl, Form B, Form B-HCl, or any combination thereofdescribed herein. In preferred embodiments, the kit is used to measurethe density of CFTR.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES

Methods & Materials

XRPD (X-Ray Powder Diffraction)

Instrument 1

X-ray powder diffraction (XRPD) data are recorded at room temperatureusing a Rigaku/MSC MiniFlex Desktop Powder X-ray Diffractometer (Rigaku,The Woodlands, Tex.). The X-Ray is generated using Cu tube operated at30 kV and 15 mA with Kβ suppression filter. The divergence slit isvariable with the scattering and receiving slits set at 4.2 degree andslit 0.3 mm, respectively. The scan mode is fixed time (FT) with 0.02degree step width and count time of 2.0 seconds. The Powder X-rayDiffractometer is calibrated using reference standard: 75% Sodalite(Na₃Al₄Si₄O₁₂Cl) and 25% Silicon (Rigaku, Cat# 2100/ALS). The sixsamples stage is used with zero background sample holders(SH-LBSI511-RNDB). The powder sample is placed on the indented area andflattened with glass slide.

Instrument 2

Alternatively, the powder x-ray diffraction measurements were performedusing PANalytical's X-pert Pro diffractometer at room temperature withcopper radiation (1.54060 A). The incident beam optic was comprised of avariable divergence slit to ensure a constant illuminated length on thesample and on the diffracted beam side. A fast linear solid statedetector was used with an active length of 2.12 degrees 2 theta measuredin a scanning mode. The powder sample was packed on the indented area ofa zero background silicon holder and spinning was performed to achievebetter statistics. A symmetrical scan was measured from 4-40 degrees 2theta with a step size of 0.017 degrees and a scan step time of 15.5 s.

Instrument 3

Alternatively, high resolution data were collected at room temperatureat the beamline ID31 (European Synchrotron Radiation Facility inGrenoble, France) The X-rays are produced by three 11-mm-gap ex-vacuumundulators. The beam is monochromated by a cryogenically cooleddouble-crystal monochromator (Si(111) crystals). Water-cooled slitsdefine the size of the beam incident on the monochromator, and of themonochromatic beam transmitted to the sample in the range of 0.5 to 2.5mm (horizontal) by 0.1 to 1.5 mm (vertical). The wavelength used for theexperiment was 1.29984 (3) Å. The diffractometer consists of a bank ofnine detectors which is scanned vertically to measure the diffractedintensity as a function of 2θ. Each detector is preceded by a Si(111)analyser crystal and the detector channels are approximately 2° apart.This diffractometer is capable of producing very precise high resolutiondiffraction patterns with peak widths as low as 0.003°, and accuracy ofpeak positions is in the order of 0.0001°. The powder diffraction datawere processed and indexed using Materials Studio (Reflex module). Thestructure was solved using PowderSolve module of Materials Studio. Theresulting solution was assessed for structural viability andsubsequently refined using Rietveld refinement procedure.

The XPRD spectra described in the examples for Form A and Form B wererecorded using Instrument 1 (FIG. 10A) or Instrument 2 (FIG. 10B) withthe settings described above. XPRD spectra described in the examples forForm B-HCl, and Form A-HCl were recorded using Instrument 2 with thesettings described above. The crystal system, space group and unit celldimensions for Form A-HCl and Form B-HCl were determined usinginstrument 3.

Differential Scanning calorimetry (DSC)

Differential Scanning calorimetry (DSC) was performed using TA DSC Q2000differential scanning calorimeter (TA Instruments, New Castle, Del.).The instrument was calibrated with indium. Samples of approximately 2-3mg were weighed into hermetic pans that were crimped using lids with onehole. The DSC samples were scanned from 25° C. to 315° C. at a heatingrate of 10° C./min. Data was collected by Thermal Advantage Q Series™software and analyzed by Universal Analysis software (TA Instruments,New Castle, Del.).

Thermogravimetric Analysis (TGA)

Thermogravimetric Analysis (TGA) data were collected on a TA Q500Thermogravimetric Analyzer (TA Instruments, New Castle, Del.). A samplewith weight of approximately 3-5 mg was scanned from 25° C. to 350° C.at a heating rate of 10° C./min. Data were collected by ThermalAdvantage Q Series™ software and analyzed by Universal Analysis software(TA Instruments, New Castle, Del.).

FTIR Spectroscopy

FTIR spectra were collected from a Thermo Scientific, Nicolet 6700 FT-IRspectometer, with smart orbit sampling compartment (multi-bounceAttenuated Total Reflection accessory), diamond window at 45 degrees.The Software used for data collection and analysis is: Omnic, 7.4. Thecollection settings were as follows:

-   -   Detector: DTGS KBr;    -   Beamsplitter: Ge on KBr;    -   Source: EverGlo IR;    -   Scan range:4000-400 cm⁻¹;    -   Gain:8.0;    -   Optical velocity:0.6329 cm/sec;    -   Aperture:100;    -   No. of scans:32; and    -   Resolution:4 cm⁻¹

The powder sample was placed directly on the diamond crystal andpressure was added to conform the surface of the sample to the surfaceof the diamond crystal. The background spectrum was collected and thenthe sample spectrum was collected.

Solid State Nuclear Magnetic Spectroscopy

Solid state nuclear magnetic spectroscopy (SSNMR) spectra were acquiredon Bruker 400 MHz proton frequency wide bore spectrometer. Protonrelaxation longitudinal relaxation times (¹H T₁) were obtained byfitting proton detected proton saturation recovery data to anexponential function. These values were used to set an optimal recycledelay of carbon cross-polarization magic angle spinning experiment (¹³CCPMAS), which, typically, was set between 1.2×1H T₁ and 1.5×¹H T₁. Thecarbon spectra were acquired with 2 ms contact time using linearamplitude ramp on proton channel (from 50% to 100%) and 100 kHz TPPMdecoupling. The typical magic angle spinning (MAS) speed was 15.0 kHz.Fluorine spectra were obtained using proton decoupled, directpolarization MAS experiment. 100 kHz TPPM decoupling was used. Therecycle delay was set to ≧5×¹⁹F T₁. The fluorine longitudinal relaxationtime (¹⁹F T₁) was obtained by fitting fluorine detected, protondecoupled saturation recovery data to an exponential function. Carbon aswell as fluorine spectra were externally referenced using the upfieldresonance of solid phase adamantane which was set to 29.5 ppm. Usingthis procedure, carbon spectra were indirectly referenced to tetramethylsilane at 0 ppm and fluorine spectra were indirectly referenced tonitromethane at 0 ppm.

Synthetic Examples Preparative Example 7-azabicyclo[2.2.1]heptanehydrochloride (8-HCl)

Preparation of trans-4-(tert-butoxycarbonylamino)cyclohexanol (A),method 1. Sodium carbonate (920.2 g, 8.682 mol, 2 eq) was added to areaction vessel followed by an addition of water (3.000 L, 6 vol) andstirring. Dichloromethane (DCM, 4.000 L, 4 vol) was added followed bytrans-4-aminocyclohexanol (500.0 g, 4.341 mol) to generate a biphasicreaction mixture that was vigorously stirred at room temperature. Asolution of Boc₂O (947.4 g, 997.3 mL, 4.341 mol, 1 eq) in DCM (2 vol)was then rapidly added dropwise to the vessel, and the resultingreaction mixture was stirred at room temperature overnight. The reactionmixture was then filtered and the filter cake was washed with water (2×8vol). The product was suction-dried until it was a compact cake. Thecake was then dried in a vacuum oven at 35° C. for 24 h giving 830 g oftrans-4-(tert-butoxycarbonylamino)cyclohexanol (A) as a crystallinesolid.

Preparation of trans-4-(tert-butoxycarbonylamino)cyclohexanol (A),method 2. Two 50 L three-neck round bottom flasks were each equippedwith a mechanical stirrer and thermocouple. The flasks were placed in acooling tub, and then each flask was charged with water (8.87 L) andtrans-4-aminocyclohexanol (1479 g). After about 10 to 30 minutes, thetrans-4-aminocyclohexanol had dissolved, and potassium carbonate (1774.6g) was added to each flask. After about 10 to 20 minutes, the potassiumcarbonate had dissolved, and DCM (2.96 L) was charged to each flask. Bocanhydride (3082.6 g) in DCM (1479 mL) was then added to each flask atsuch a rate as to maintain the temperature at 20 to 30° C. An ice/waterbath was used to control the exotherm and to accelerate the addition,which took approximately 1 to 2 hours. A suspension formed during theaddition, and the reaction mixtures were allowed to warm to roomtemperature and stirred overnight, until the reaction was complete basedon the disappearance of the Boc anhydride. Heptane (6 L) was thencharged to each flask, and the mixtures were cooled to approximately 0to 5° C. Solids were collected from each flask by filtration using thesame filter. The combined solids were washed with heptane (6 L) followedby water (8 L). The solids were charged to an appropriately sized crockequipped with a mechanical stirrer. Water (12 L) and heptane (6 L) wereadded, and the resulting suspension was mechanically stirred for 30 to60 minutes. The solids were collected by filtration and then washed on afilter with water (8 L) and heptane (8 L), air-dried on a filter forthree days, and then dried under vacuum at 30 to 35° C. to a constantweight to provide the product as a white solid.

Preparation oftrans-4-(tert-butoxycarbonylamino)cyclohexylmethanesulfonate (B),method 1. A 12 L flask was equipped with a nitrogen flow and amechanical stirrer. Trans-4-(tert-butoxycarbonylamino)cyclohexanol (750g, 3.484 mol) was introduced, followed by tetrahydrofuran (THF, 6.000 L,8 vol), and the mixture was stirred. Triethylamine (370.2 g, 509.9 mL,3.658 mol, 1.05 eq) was added and the mixture was cooled to 0° C.Methanesulfonyl chloride (419.0 g, 283.1 mL, 3.658 mol, 1.05 eq) wascarefully added dropwise, keeping the temperature of the mixture below5° C. After the addition, the mixture was stirred at 0° C. for 3 h, andthen gradually warmed to room temperature (17° C.) and stirred overnight(about 15 h). The mixture was quenched with water (6 vol) and stirredfor 15 min. Ethyl acetate (EtOAc, 9.000 L, 12 vol) was added and thestirring was continued for 15 min. The stirring was stopped and themixture was allowed to stand for 10 min, and the aqueous phase wasremoved. 1 N HCl (6 vol, 4.5 L) was added and stirring was continued for15 min. The stirring stopped and the aqueous phase was removed. 10% w/vNaHCO₃ (4.5 L, 6 vol) was added and the mixture stirred for 10 min.Stirring was stopped and the aqueous phase was removed. Water (6 vol,4.5 L) was added and the mixture was stirred for 10 min. The aqueouslayer was removed, and the organic layer was polish filtered andconcentrated to 4 vol. Heptane (5.5 vol, 4 L) was added and the mixturewas concentrated again to dryness resulting in 988 g oftrans-4-(tert-butoxycarbonylamino)cyclohexylmethanesulfonate.

Preparation oftrans-4-(tert-butoxycarbonylamino)cyclohexylmethanesulfonate (B), method2. A three-neck round bottom flask equipped with a mechanical stirrer,addition funnel, nitrogen inlet, thermocouple and drying tube was placedinto a cooling tub. Trans-4-(tert-butoxycarbonylamino)cyclohexanol (2599g, 12.07 mol, 1.0 eq), tetrahydrofuran (THF) (20.8 L), and triethylamine(1466 g, 14.49 mol, 1.2 eq) were added to the flask. The mixture wascooled with an ice water bath and stirred. Methanesulfonyl chloride(1466 g, 12.80 mol, 1.06 eq) was added dropwise by addition funnel over1 hour. Once the addition was complete, the cooling bath was removed,and the reaction mixture was stirred until TLC indicated the startingmaterial was consumed (about 30 minutes). The reaction mixture was thenquenched with an aqueous solution of hydrochloric acid (223 mL of HCl in6.7 L of water) and EtOAc (10.4 L). The mixture was stirred forapproximately 10 to 20 minutes at ambient temperature and then wastransferred to a separatory funnel. The layers were separated, and theaqueous layer discarded. The organic layer was washed with water (2×4.5L), aqueous saturated sodium bicarbonate solution (1×4.5 L), and driedover anhydrous magnesium sulfate with stirring for 5 to 10 minutes. Themixture was filtered and the filter cake was washed with EtOAc (2×600mL). The combined washes and filtrate were concentrated under reducedpressure at 40° C., leaving a white solid. The solid was taken up inheptane (3 L) and cooled in an ice/methanol cooling tub. More heptane (5L) was added, and the mixture was stirred at 0 to 5° C. for not lessthan 1 hour. The solids were then collected by filtration, washed withcold heptane (0 to 5° C., 2×1.3 L), and dried under vacuum at 40° C. toa constant weight to provide the captioned compound.

Note: A jacketed reactor may be used instead of a round bottom flaskwith a cooling tub and ice bath.

Preparation of trans-4-aminocyclohexylmethanesulfonate (C), method 1.Trans-4-(tert-butoxycarbonylamino)cyclohexylmethanesulfonate (985 g,3.357 mol) was introduced into a 3-neck 12 L flask equipped with astirrer under a nitrogen atmosphere and open vent. DCM (1.970 L, 2 vol)was added at room temperature, and stirring was commenced.Trifluoroacetic acid (TFA) (2.844 kg, 1.922 L, 24.94 mol, 2 vol) wasslowly added to the mixture in two batches of 1 L each. After the firstaddition, the mixture was stirred for 30 min followed by a secondaddition. The mixture was stirred overnight (15 h) at room temperatureresulting in a clear solution. 2-methyltetrahydrofuran (4 vol) was thenadded to the reaction mixture, which was stirred for 1 h. The mixturewas then carefully filtered in a fume hood and suction dried to generate1100 g of TFA salt of trans-4-aminocyclohexylmethanesulfonate withexcess TFA.

Preparation of trans-4-aminocyclohexylmethanesulfonate (C), method 2. A50 L three-neck round bottom flask was equipped with a mechanicalstirrer, addition funnel and thermocouple and was placed into a coolingtub. To the flask was addedtrans-4-(tert-butoxycarbonylamino)cyclohexylmethanesulfonate (3474 g,1.0 eq) and DCM (5.9 L) to the flask. The resulting suspension wasstirred for 5 to 10 minutes at ambient temperature, and thentrifluoroacetic acid (TFA, 5.9 L) was added via addition funnel slowlyover 2.5 hours to control the resulting exotherm and rate of gasevolution. The reaction mixture was stirred at room temperatureovernight and then cooled to 15° C. to 20° C. using an ice water bath.2-Methyl tetrahydrofuran (2-MeTHF, 11.8 L) was then added via theaddition funnel at a rate to maintain the internal temperature below 25°C. (approximately 1.5 hours). The addition of the first 4-5 L of 2-MeTHFwas exothermic. The resulting suspension was stirred for 1 hour. Thesolids were collected by filtration and then washed with 2-MeTHF (2×2.2L) and then dried under vacuum at ambient temperature to a constantweight to provide the captioned compound as a white solid.

Preparation of 7-azabicyclo[2.2.1]heptane hydrochloride (8-HCl),method 1. The TFA salt of trans-4-aminocyclohexylmethanesulfonate (200g, 650.9 mmol) was introduced into a 3-necked flask followed by theaddition of water (2.200 L, 11 vol). NaOH (78.11 g, 1.953 mol, 3 eq) wasslowly added, keeping the temperature of the reaction mixture below 25°C. and the mixture was stirred overnight. DCM (1.4 L, 7 vol) was thenadded and the mixture stirred, and the organic layer was separated. Theaqueous layer was then extracted a second time with DCM (1.4 L, 7 vol),and the DCM layers were combined. HCl (108.5 mL, 12M, 1.3020 mol, 2 eq)was then added, the mixture was stirred for 30 min and then concentratedon a rotary evaporator to dryness. Acetonitrile (10 vol) was added andthe mixture concentrated. This was repeated 3 times until all tracewater was azeotropically removed, to provide 7-azabicyclo[2.2.1]heptanehydrochloride. The crude product was recrystallized from acetonitrile(10 vol) to provide 7-azabicyclo[2.2.1]heptane hydrochloride 8-HCl as acolorless crystalline solid. ¹HNMR (DMSO-d⁶) ppm 8.02-8.04 (d);7.23-7.31 (m); 4.59 (s); 3.31 (s); 2.51-3.3 (m); 1.63-1.75 (m);1.45-1.62 (m).

As a note, instead of adding DCM for extraction, the crude product canalso be distilled at about 95° C. to 97° C. and further recrystallized.

Preparation of 7-azabicyclo[2.2.1]heptane hydrochloride (8-HCl), method2. A 50 L three neck round bottom flask equipped with a mechanicalstirrer, addition funnel and thermocouple and was placed into a heatingmantle. Trans-4-aminocyclohexylmethanesulfonate trifluoroacetate in(3000 g, 1 eq) and water (30 L) were added to the flask. The mixture wasstirred, as 50% NaOH (2343 g, 29.29 mol, 3 eq) was added by an additionfunnel at such a rate as to maintain the temperature below 25° C.because the addition was mildly exothermic. Upon completion of the NaOHaddition, the reaction mixture was stirred overnight at roomtemperature. The product was recovered by fractional distillation atreflux temperature, (approximately 100° C.) with a head temperature of95° C. to 98° C. The pH of each fraction was adjusted to 2 by addingHCl, and concentrated under reduced pressure at 55° C. to leave a thickpaste. Acetonitrile (ACN 1.5 L) was added and the resulting suspensionwas stirred for 30 minutes and then cooled to 0° C. to 5° C. for 1 hour.The solids were collected by filtration, washed with cold (0 to 5° C.)ACN (2×600 mL), and dried under vacuum at 50° C. to a constant weight.

A 22 L three-neck round bottom flask was equipped with a mechanicalstirrer, thermocouple, and condenser and placed into a heating mantle.The collected solids (2382 g), methanol (4.7 L) and 2-MeTHF (4.7 L) wereadded to the flask. The resulting suspension was stirred and heated toreflux (approximately 65° C.). The reaction flask was transferred to acooling tub, and the mixture was stirred. 2-MeTHF (4.7 L) was then addedvia addition funnel over 30 minutes. The resulting suspension was cooledto 0 to 5° C. and stirred at this temperature for 30 minutes. The solidswere collected by filtration, washed with cold (0 to 5° C.) 2-MeTHF(2×600 mL), and then dried under vacuum at 55° C. to a constant weight.

A 12 L three-neck round bottom flask equipped with a mechanical stirrer,thermocouple, nitrogen inlet and condenser was placed into a heatingmantle. The crude product (2079 g) and ACN (6.2 L) were added to theflask. The resulting suspension was stirred and heated to reflux(approximately 82° C.) for 30 minutes. The flask was transferred to acooling tub and the suspension was slowly cooled to 0 to 5° C. andmaintained at this temperature for 1 hour. The solids were collected byfiltration, washed with cold (0 to 5° C.) ACN (3×600 mL), and driedunder vacuum at 55° C. to a constant weight affording to provide thecaptioned product.

Example 1A Preparation of4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid (2)

Preparation of diethyl2-((2-chloro-5-(trifluoromethyl)phenylamino)methylene)malonate (6B).2-Chloro-5-(trifluoromethyl)aniline 4 (200 g, 1.023 mol), diethyl2-(ethoxymethylene)malonate (5) (276 g, 1.3 mol) and toluene (100 mL)were combined under a nitrogen atmosphere in a 3-neck, 1-L round bottomflask equipped with Dean-Stark condenser. The solution was heated withstirring to 140° C. and the temperature was maintained for 4 h. Thereaction mixture was cooled to 70° C. and hexane (600 mL) was slowlyadded. The resulting slurry was stirred and allowed to cool to roomtemperature. The solid was collected by filtration, washed with 10%ethyl acetate in hexane (2×400 mL) and then dried under vacuum toprovide a white solid (350 g, 94% yield) as the desired condensationproduct diethyl2-((2-chloro-5-(trifluoromethyl)phenylamino)methylene)malonate (6B). ¹HNMR (400 MHz, DMSO-d₆) δ 11.28 (d, J=13.0 Hz, 1H), 8.63 (d, J=13.0 Hz,1H), 8.10 (s, 1H), 7.80 (d, J=8.3 Hz, 1H), 7.50 (dd, J=1.5, 8.4 Hz, 1H),4.24 (q, J=7.1 Hz, 2H), 4.17 (q, J=7.1 Hz, 2 H), 1.27 (m, 6H).

Preparation of ethyl8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate(6A) Method 1. A 3-neck, 1 L flask was charged with Dowtherm® (200 mL, 8mL/g), which was degassed at 200° C. for 1 h. The solvent was heated to260° C. and charged in portions over 10 min with diethyl2-((2-chloro-5-(trifluoromethyl)phenylamino)methylene)malonate (6B) (25g, 0.07 mol). The resulting mixture was stirred at 260° C. for 6.5 hours(h) and the resulting ethanol byproduct removed by distillation. Themixture was allowed to slowly cool to 80° C. Hexane (150 mL) was slowlyadded over 30 minutes (min), followed by an additional 200 mL of hexaneadded in one portion. The slurry was stirred until it had reached roomtemperature. The solid was filtered, washed with hexane (3×150 mL), andthen dried under vacuum to provide ethyl8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate(6A) as a tan solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.91 (s, 1H), 8.39 (s,1H), 8.06 (d, J=8.3 Hz, 1H), 7.81 (d, J=8.4 Hz, 1H), 4.24 (q, J=7.1 Hz,2H), 1.29 (t, J=7.1 Hz, 3H).

Preparation of ethyl8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate(6A) Method 2. Compound 6B (2000 g, 5.468 mol) was introduced into thereactor. Dowtherm (4.000 L) was charged to the reactor and degassed atroom temperature overnight with nitrogen purge. It was then stirred andwarmed to 260° C. EtOH produced was distilled off. The reaction wasmonitored and was complete after 5.5 h, the reaction was substantiallycomplete. Heat source was removed and the reaction mixture was cooled to80° C. and heptane (2.000 L) was charged. The mixture was stirred for 30min. Heptane (6.000 L) was charged to the stirred mixture and stirringcontinued overnight. Solids were filtered off and washed with heptane(4.000 L) and dried in a vacuum oven at 50° C. to provide Compound 6A.

Preparation of ethyl4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylate (6C). A 3-neck, 5 Lflask was charged with of ethyl8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate(6A) (100 g, 0.3 mol), ethanol (1250 mL, 12.5 mL/g) and triethylamine(220 mL, 1.6 mol). The vessel was then charged with 10 g of 10% Pd/C(50% wet) at 5° C. The reaction was stirred vigorously under hydrogenatmosphere for 20 h at 5° C., after which time the reaction mixture wasconcentrated to a volume of approximately 150 mL. The product, ethyl4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylate (6C), as a slurrywith Pd/C, was taken directly into the next step.

Preparation of4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid (2).Ethyl 4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylate (6C) (58 g,0.2 mol, crude reaction slurry containing Pd/C) was suspended in NaOH(814 mL of 5 M, 4.1 mol) in a 1 L flask with a reflux condenser andheated at 80° C. for 18 h, followed by further heating at 100° C. for 5h. The reaction was filtered warm through packed Celite to remove Pd/Cand the Celite was rinsed with 1 N NaOH. The filtrate was acidified toabout pH 1 to obtain a thick, white precipitate. The precipitate wasfiltered then rinsed with water and cold acetonitrile. The solid wasthen dried under vacuum to provide4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid (2) asa white solid. ¹H NMR (400.0 MHz, DMSO-d₆) δ 15.26 (s, 1H), 13.66 (s,1H), 8.98 (s, 1H), 8.13 (dd, J=1.6, 7.8 Hz, 1H), 8.06-7.99 (m, 2H).

Example 1B Alternative Preparation of4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid (2)

Preparation of8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylicacid (6D). Ethyl8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate(6B) (1200 g, 3.754 mol) was charged into a vessel followed by theaddition of 2-propanol (1.200 L) and water (7.200 L) and stirred. NaOH(600.6 g, 7.508 mol) and water (1.200 L) were mixed and allowed to coolto room temperature. The resulting NaOH solution was charged into thereaction vessel. The reaction mixture was heated to 80° C. and stirredfor 3.5 h generating a dark and homogenous mixture. After an additionalhour, acetic acid (9.599 L [of a 20% w/v solution], 31.97 mol) was addedvia dropping funnel over 45 min. The reaction mixture was cooled to 22°C. at a rate of 6° C./h with stirring. The resulting solid was filtered,washed with water (3 L) to generate a wet cake of (1436 g). The filtratewas dried in a vacuum oven with nitrogen bleed over Drierite® togenerate8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylicacid as a brown solid. The8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylicacid was purified by slurrying in 1.5 L methanol, with stirring, for 6h. It was then filtered and dried to furnish 968.8 g of purified8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylicacid (6D).

Preparation of4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid

(2).8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylicacid (6D) (18.5 g, 1.00 eq, limiting reagent) was charged into areaction vessel and MeOH (118 mL, 6.4 vol) was added under inertatmosphere with agitation. NaOMe (3.53 g, 1.00 eq.) was added portionwise over 10 min to the reactor. The mixture was stirred until allsolids were in solution (5-10 minutes). Palladium on carbon (2.7 g, 0.03eq) was then added to the reaction mixture. Potassium formate (10.78 g,2 eq.) dissolved in MeOH (67 mL, 3.6 vol) was added to the reactionmixture over 30 min. It was then stirred for about 4.5 h at ambienttemperature. The reaction was judged complete when8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylicacid is no more than 1.0% with respect to4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid. Whenthe reaction was complete, the mixture was filtered through a pad ofCelite (37 g [˜approximately twice the mass of the starting material,6D]) to remove solids. The Celite cake was washed with MeOH (37 mL, 2vol). The filtrate was charged into a clean reaction vessel and stirred.Acetic acid (7.22 mL, 2 eq.) was charged continuously to the stirredsolution over at least 45 minutes and the resulting slurry stirred forbetween 5-16 h. The solid was filtered and the cake washed with MeOH (56mL, 3 vol), suction-dried and then vacuum dried to provide the captionedcarboxylic acid 2.

Alternatively, the potassium formate reagent may be replaced withhydrogen gas.

Example 2A Preparation of4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (3)

Preparation of7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (9),method 1. To a flask containing 7-azabicyclo[2.2.1]heptane hydrochloride(8-HCl) (4.6 g, 34.43 mmol, obtained from under a nitrogen atmospherewas added a solution of 4-fluoro-1-nitro-2-(trifluoromethyl)benzene (7)(6.0 g, 28.69 mmol) and triethylamine (8.7 g, 12.00 ml, 86.07 mmol) inacetonitrile (50 ml). The reaction flask was heated at 80° C. under anitrogen atmosphere for 16 h. The reaction mixture was allowed to cooland then was partitioned between water and dichloromethane. The organiclayer was washed with 1 M HCl, dried over Na₂SO₄, filtered, andconcentrated to dryness. Purification by silica gel chromatography(0-10% ethyl acetate in hexanes) yielded7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (9) asa yellow solid. ¹H NMR (400.0 MHz, DMSO-d₆) δ 8.03 (d, J=9.1 Hz, 1H),7.31 (d, J=2.4 Hz, 1H), 7.25 (dd, J=2.6, 9.1 Hz, 1 H), 4.59 (s, 2H),1.69-1.67 (m, 4H), 1.50 (d, J=7.0 Hz, 4H).

Preparation of7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (9),method 2. 4-fluoro-1-nitro-2-(trifluoromethyl)benzene (7) (901 g, 4.309mol) was introduced into a 30 L jacketed vessel along with Na₂CO₃ (959.1g, 9.049 mol) and DMSO (5 L, 5.5 vol) under nitrogen atmosphere andstirring. 7-azabicyclo[2.2.1]heptane hydrochloride (8-HCl) (633.4 g,4.740 mol) was then added to the vessel in portions. The temperature wasgradually raised to 55° C. When the reaction was substantially complete,the mixture was diluted with 10 vol EtOAc and washed with water (5.5vol) three times or until DMSO in the aqueous layer disappeared (HPLC).The organic layer was concentrated to 4 vol and then the solvent wasswapped with cyclohexane until all the EtOAc was removed, and the totalvolume in the flask was about 4 vol containing cyclohexane. The reactionmixture was heated to 60° C. on a rotary evaporator for 30 min. Then thesolution was cooled to room temperature with stirring or rotation for 3h. When all the solid crystallized, the solution was concentrated todryness to provide7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (9).

Preparation of7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (9),method 3. 4-fluoro-1-nitro-2-(trifluoromethyl)benzene was dissolved in 3vol DCM. Tetrabutylammoniumbromide (0.05 eq) and KOH (50 wt %, 3.6 eq)were added. 7-azabicyclo[2.2.1]heptane hydrochloride (8-HCl) was thenadded at 0-5° C. The reaction was warmed up to ambient temperature andmonitored by HPLC. Once substantially complete, the layers wereseparated and the organic layer was washed with 1M HCl. The layers wereseparated and the aqueous layer was discarded. The organic layer waswashed once with water, once with brine, and then distilled. Theresulting material was recrystallized from cyclohexane at reflux. Thesolid was filtered, washed with cyclohexane, and dried in a vacuum ovenat 45° C. with a N₂ gas bleed to provide7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (9).

Preparation of4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (3). Aflask charged with7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (26)(7.07 g, 24.70 mmol) and 10% Pd/C (0.71 g, 6.64 mmol) was evacuated andthen flushed with nitrogen. Ethanol (22 ml) was added and the reactionflask was fitted with a hydrogen balloon. After stirring vigorously for12 h, the reaction mixture was purged with nitrogen and Pd/C was removedby filtration. The filtrate was concentrated to a dark oil under reducedpressure and the residue purified by silica gel chromatography (0-15%ethyl acetate in hexanes) to provide4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (3) as apurple solid (5.76 g, 91% yield). ¹H NMR (400.0 MHz, DMSO-d₆) δ 6.95(dd, J=2.3, 8.8 Hz, 1H), 6.79 (d, J=2.6 Hz, 1H), 6.72 (d, J=8.8 Hz, 1H),4.89 (s, 2H), 4.09 (s, 2H), 1.61-1.59 (m, 4H) and 1.35 (d, J=6.8 Hz,4H).

Example 2B Preparation of the hydrochloride salt of447-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (3-HCl)

Preparation of the hydrochloride salt of4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (3-HCl),method 1. Palladium on carbon (150 g, 5% w/w) was charged into a BüchiHydrogenator (20 L capacity) under a nitrogen atmosphere followed by theaddition of the hydrochloride salt of7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (9)(1500 g) and 2-methyltetrahydrofuran (10.5 L, 7 vol). Hydrogen gas wascharged into the closed vessel to a pressure of +0.5 bar aboveatmospheric pressure. A vacuum was applied for about 2 min followed bythe introduction of hydrogen gas to a pressure of 0.5 bar. This processwas repeated 2 times. Then hydrogen gas was continuously charged at +0.5bar above atmospheric pressure. The mixture was stirred and thetemperature was maintained between 18° C. and 23° C. by cooling thejacket of the vessel. Once the reaction consumed no more hydrogen andevolved no more heat, a vacuum was again applied. Nitrogen gas wascharged into the vessel at 0.5 bar and a vacuum was reapplied followedby a second charge of 0.5 bar nitrogen gas. When the reaction wassubstantially complete, the reaction mixture was transferred into areceiving flask under nitrogen atmosphere via a filter funnel using aCelite filter. The Celite filter cake was washed with2-methyltetrahydrofuran (3 L, 2 vol). The washings and filtrate werecharged into a vessel equipped with stirring, temperature control, and anitrogen atmosphere. 4M HCl in 1,4-dioxane (1 vol) was addedcontinuously over 1 h into the vessel at 20° C. The mixture was stirredfor an additional 10 h (or overnight), filtered, and washed with2-methyltetrahydrofuran (2 vol) and dried to generate 1519 g of the of4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)anilinehydrochloride (3-HCl) as a white crystalline solid.

Alternative solvents may also be substituted in this example. Forinstance, MeOH and/or EtOH could be used in place of 2-MeTHF.

Example 3A Preparation ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamideas Form A (Compound 1 Form A)

To a solution of 4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylicacid (2) (9.1 g, 35.39 mmol) and4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (3) (9.2g, 35.74 mmol) in 2-methyltetrahydrofuran (91.00 mL) was added propylphosphonic acid cyclic anhydride (50% solution in ethyl acetate, 52.68mL, 88.48 mmol) and pyridine (5.6 g, 5.73 mL, 70.78 mmol) at roomtemperature. The reaction flask heated at 65° C. for 10 h under anitrogen atmosphere. After cooling to room temperature the reaction wasthen diluted with ethyl acetate and quenched with saturated Na₂CO₃solution (50 mL). The layers were separated, and the aqueous layer wasextracted twice more with ethyl acetate. The combined organic layerswere washed with water, dried over Na₂SO₄, filtered and concentrated toa tan solid. The crude solid product was slurried in ethylacetate/diethyl ether (2:1), collected by vacuum filtration, and washedtwice more with ethyl acetate/diethyl ether (2:1) to provide the crudeproduct as a light yellow crystalline powder. The powder was dissolvedin warm ethyl acetate and absorbed onto Celite. Purification by silicagel chromatography (0-50% ethyl acetate in dichloromethane) providedForm A ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide(Compound 1 Form A) as a white crystalline solid. LC/MS m/z 496.0[M+H]⁺, retention time 1.48 min (RP-C₁₈, 10-99% CH₃CN/0.05% TFA over 3min). ¹H NMR (400.0 MHz, DMSO-d₆) δ 13.08 (s, 1H), 12.16 (s, 1H), 8.88(s, 1H), 8.04 (dd, J=2.1, 7.4 Hz, 1H), 7.95-7.88 (m, 3H), 7.22 (dd, 2.5,8.9 Hz, 1H), 7.16 (d, J=2.5 Hz, 1H), 4.33 (s, 2H), 1.67 (d, J=6.9 Hz,4H), 1.44 (d, J=6.9 Hz, 4H).

The powder diffractogram of Compound 1 Form A is shown in FIG. 1.

Table 1 provides a list of XRPD peaks representative of Compound 1 FormA.

TABLE 1 Representative XRPD peaks of Compound 1 Form A. 2-Theta(degrees) Relative Intensity (%) 7.9 100.0 9.3 10.8 11.9 12.8 14.4 35.215.1 12.6 15.8 34.1 17.0 25.2 17.7 13.8 19.3 39.4 20.1 20.2 21.4 14.521.8 94.2 23.4 30.0 23.8 92.0 25.6 8.9 26.8 6.4 29.4 8.1 29.7 18.1 30.114.2 31.2 9.9

A representative sample of Compound 1 Form A gave the FTIR spectrumprovided in FIG. 2.

Conformational pictures of Compound 1 Form A based on single X-rayanalysis are shown in FIG. 3. Diffraction data were acquired on a BrukerApex II Diffractometer equipped with sealed tube CuK-alpha source and anApex II CCD detector. The structure was solved and refined using SHELXprogram (Sheldrick, G. M., Acta Cryst. A64, pp. 112-122 (2008)). Basedon intensities, statistics and symmetry, the structure was solved andrefined in a trigonal crystal system and an R-3 space group. Compound 1Form A has the following unit cell dimensions: a=19.1670(4) Å,b=19.1670(4) Å, c=33.6572(12) Å, α=90°, β=90°, and γ=120°.

Example 3B Preparation ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamideas Form A-HCl (Compound 1 Form A-HCl)

2-methyltetrahydrofuran (0.57 L, 1.0 vol) was charged into a 30 Ljacketed reactor vessel, followed by the addition of the hydrochloridesalt of 4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline(3-HCl) (791 g, 2.674 mol) and4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid (2)(573 g, 2.2278 mol) and an additional 5.2 L (9.0 vol) of2-methyltetrahydrofuran. Stirring commenced and T3P in2-methyltetrahydrofuran (2.836 kg, 4.456 mol) was added to the reactionmixture over 15 min. Then, pyridine (534.0 g, 546.0 mL, 6.684 mol) wasadded via an addition funnel dropwise over 30 min. The mixture waswarmed to 45° C. over about 30 min and stirred for 12-15 h. HPLCanalysis indicated that that4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid waspresent in an amount less than 2%. The mixture was then cooled to roomtemperature. 2-methyltetrahydrofuran (4 vol, 2.292 L) was added followedby water (6.9 vol, 4 L), while the temperature was maintained below 30°C. The water layer was removed and the organic layer was carefullywashed twice with NaHCO₃ saturated aqueous solution. The organic layerwas then washed with 10% w/w citric acid (5 vol) and finally with water(7 vol). The mixture was polished filtered and transferred into anotherdry vessel. Seed crystals of Form A-HCl ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamidehydrochloride (Compound 1 Form A-HCl) (3.281 g, 5.570 mmol) were added.HCl (g) (10 eq) was bubbled over 2 h and the mixture was stirredovernight. The resulting suspension was filtered, washed with2-methyltetrahydrofuran (4 vol), suction dried and oven dried at 60° C.until constant weight to provide Compound 1 Form A-HCl.

The powder diffractogram of Compound 1 Form A-HCl is shown in FIG. 4.

Table 2 provides representative XRPD peaks of Compound 1 Form A-HCl.

TABLE 2 Representative XRPD Peaks of Compound 1 Form A-HCl. 2-Theta(degrees) Relative Intensity (%) 7.1 44.3 8.2 33.3 10.8 1.5 11.7 1.512.1 5.8 13.7 3.3 14.1 32.1 14.7 16.9 15.0 5.7 16.1 3.0 16.4 16.9 16.63.7 16.8 1.9 17.6 0.6 18.7 12.4 18.9 1.9 19.7 5.4 19.8 6.9 20.3 1.5 21.2100.0 21.7 10.6 21.9 12.3 22.2 4.0 22.8 21.9 23.4 9.8 24.6 34.3 25.017.9 25.2 8.6 25.9 3.6 26.5 1.5 26.9 7.0 27.5 8.3 28.0 5.3 28.3 1.6 28.73.5 29.0 4.8 29.2 7.5 29.8 1.40 30.1 1.8 31.0 5.4 31.3 2.6 31.9 1.7 32.34.6 32.4 3.7 32.8 2.3 33.3 3.9 34.3 1.8 34.5 3.6 34.7 8.7 35.3 3.0 35.612.7 35.6 18.9 36.1 3.2 36.8 3.3 37.2 1.7 37.8 3.1 38.5 2.8 39.1 3.139.7 2.3

A single crystal of Compound 1 Form A-HCl was determined to possess amonoclinic crystal system, a P2₁/c space group, and the following unitcell dimensions: a=13.6175(4) Å, b=21.614(3) Å, c=8.3941(4) Å, α=90°,β=112.303°, and γ=90°.

A sample of Compound 1 Form A-HCl was also evaluated using microscopy.

A DSC curve for a sample of Compound 1 Form A-HCl is provided at FIG. 5and a TGA curve of a representative sample of Compound 1 Form A-HCl isprovided in FIG. 6.

A representative sample of Compound 1 Form A-HCl presented the FTIRspectrum provided in FIG. 7.

Compound 1 Form A-HCl was also analyzed using solid state ¹³C and ¹⁹FNMR. The respective NMR spectra are provided in FIGS. 8 and 9. Severalpeaks found in the ¹³C SSNMR and ¹⁹F SSNMR spectra are described inTables 3 and 4.

TABLE 3 ¹³C SSNMR Peaks for Compound 1 Form A-HCl. Peak No. F1 (ppm) 1175.7 2 163.7 3 142.6 4 140.8 5 137.2 6 131.5 7 129.0 8 126.0 9 124.8 10123.8 11 121.5 12 117.8 13 112.4 14 65.7 15 29.2 16 28.3 17 26.1

TABLE 4 ¹⁹F SSNMR Peaks for Compound 1 Form A-HCl. Peak No. F1 (ppm) 1−57.0 2 −60.5

Example 4A Preparation ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamideas Form B (Compound 1 Form B)

2-Methyltetrahydrofuran (1 vol) was charged into a 30 L jacketed reactorvessel followed by the addition of the hydrochloride salt of4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (3-HCl)(1.2 eq) and 4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylicacid (2) (573 g, 2.228 mol). Additional 2-methyltetrahydrofuran (9 vol)was charged into the vessel and stirring commenced. T3P in2-methyltetrahydrofuran (2 eq) was added to the reaction mixture over aperiod of 15 min. Pyridine (3 eq) was added rapidly in a dropwisefashion using an addition funnel. Under stirring, the mixture was thenheated to 45° C. over a period of about 30 min and this temperature wasmaintained for about 5 h. The mixture was cooled to room temperature.2-methyltetrahydrofuran (4 vol) was added followed by the slow additionof water (6.9 vol), and the temperature of the reaction was kept below30° C. The water layer was removed and the organic layer was washedtwice with NaHCO₃ saturated aqueous solution. The organic layer was thencarefully washed with 10% w/w citric acid (5 vol) and washed with water(7 vol), and polished filtered and transferred into another dry vessel.2-methyltetrahydrofuran (10 vol) was added and stirring commenced.Heptane (10 vol) was rapidly added in a dropwise fashion with stirring.The mixture was stirred for a period of about 12 h, and then vacuumfiltered. The solid filter cake was introduced into another vessel.Water (15 vol) was charged into the vessel and the suspension wasstirred vigorously for 48 h, then filtered. The solid cake was washedwith water (5 vol) and dried at 45° C. to constant weight to produceCompound 1 Form B.

The powder diffractograms of Compound 1 Form B recorded usinginstruments 1 and 2 are shown in FIGS. 10A and 10B respectively.

Table 5 includes the representative XRPD peaks of Compound 1 Form B

TABLE 5 Representative XRPD Peaks of Compound 1 Form B 2 Theta (degrees)Relative Intensity (%) 6.7 36.6 9.4 37.2 10.0 29.5 11.2 35.3 13.4 70.614.8 18.1 15.2 88.8 15.4 16.6 17.2 49.5 17.8 48.0 18.1 83.8 18.8 13.619.2 47.6 20.1 68.9 21.2 71.8 22.0 42.6 22.6 7.6 23.5 18.1 24.0 100.025.0 9.6 25.9 9.7 26.3 44.8 26.9 26.3 27.2 86.7 27.7 37.8 28.2 7.4 28.949.0 29.6 21.3 30.3 8.6 30.6 5.5 31.2 19.3 32.3 5.5 33.7 11.4 34.2 8.934.7 12.4 35.1 8.0 36.7 6.5 38.1 4.7 39.3 5.3

A DSC curve for a sample of Compound 1 Form B is provided at FIG. 11,and a TGA curve for a representative sample of Compound 1 Form B isprovided in FIG. 12.

A representative sample of Compound 1 Form B presented the FTIR spectrumprovided in FIG. 13.

Compound 1 Form B was also analyzed using solid state ¹³C and ¹⁹F NMR.The respective NMR spectra are provided in FIGS. 14 and 15. Severalpeaks found in the ¹³C SSNMR and ¹⁹F SSNMR spectra are described inTables 6 and 7.

TABLE 6 ¹³C SSNMR Peaks for Compound 1 Form B. Peak No. F1 (ppm) 1 175.32 165.3 3 145.9 4 141.4 5 132.9 6 126.8 7 123.5 8 117.4 9 113.4 10 58.311 29.2 12 26.9

TABLE 7 ¹⁹F SSNMR Peaks for Compound 1 Form B. Peak No. F1 (ppm) 1 −56.12 −62.1

A single crystal of Compound 1 Form B was mounted on a MicroMount loopand centered on a Broker Apex II diffractometer that was equipped with asealed copper X-ray tube and Apex II CCD detector. Initially, 3 sets of40 frames were collected to determine a preliminary unit cell.Subsequently a full data set consisting of 15 scans and 6084 frames wasacquired. Data collection was performed at room temperature. Data wereintegrated and scaled using Apex II software from Bruker AXS.Integration and scaling resulted in 6176 reflections, 2250 of which wereunique. Structure was solved by direct methods in space group P21/cusing SHELXTL software. Refinement was performed with full-matrixleast-square method on F2 using SHELXTL software as well. Altogether 392parameters were used in refinement resulting in reflection to parameterratio of 5.74. The final refinement index was wR2=0.0962 and R1=0.0682(wR2=0.0850 and R1=0.0412 for reflections with I>2 sigma(I).

The single crystal ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamidein Compound 1 Form B was determined to possess a monoclinic crystalsystem, a P21/c space group, and the following unit cell dimensions:a=13.5429(4) Å, b=13.4557(4) Å, c=12.0592(4) Å, α=90°, β=101.193°, andγ=90°.

Example 4B Preparation ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamideas Form B-HCl (Compound 1 Form B-HCl)

100 mL of 2-methyltetrahydrofuran was charged into a 3-necked flaskhaving a nitrogen atmosphere equipped with a stirrer. Compound 1 FormA-HCl (55 g, 0.103 mol) was added to the flask followed by 349 mL of2-methyltetrahydrofuran, and stirring commenced. 28 mL of water wasadded into the flask and the flask was warmed to an internal temperatureof 60° C. and stirred for 48 h. The flask was cooled to room temperatureand stirred for 1 h. The reaction mixture was vacuum filtered until thefilter cake was dry. The solid filter cake was washed with2-methyltetrahydrofuran (4 vol) twice. The solid filter cake remainedunder vacuum suction for a period of about 30 minutes and wastransferred to a drying tray. The filter cake was dried to a constantweight under vacuum at 60° C. This resulted in the generation ofCompound 1 Form B-HCl as a white crystalline solid.

Method 2.

Compound 1 Form A-HCl (14.638 g, 27.52 mmol) was charged to a 100 mLround bottom flask. EtOH (248.9 mL) and water (27.82 mL) were added. Thewhite slurry was heated to reflux. A clear solution was obtained at 77°C. The reaction was cooled to 45° C., and was allowed to stir for 30min, and then was cooled to 20° C. The mixture was allowed to stir foran additional 3 h at 20° C. The product was filtered and the cake washedwith EtOH. The solid was dried in a vacuum oven at 45° C. with anitrogen bleed to provide Compound 1 Form B-HCl as a white solid.

As a note, other solvent combination such as MeOH/H₂O and IPA/H₂O or thelike can be used instead of EtOH/H₂O as described in this example.

Examples of alternative solvent combinations are provided in Table 8.

TABLE 8 Other Solvents that Can Be Used to Make Form B-HCl. SolventSolvent Volume T [° C.] MeOH 10 60 MeOH:H₂O 10:0.2 60 MeOH:H₂O 10:0.5 60MeOH:H₂O 10:1   60 MeOH:H₂O 10:1.5 60 IPA:H₂O 10:1   75 IPA:H₂O 10:1.575 MeOH:H₂O 10:1   65 EtOH 10 70 EtOH:H₂O 10:0.2 70 EtOH:H₂O 10:0.5 70EtOH:H₂O 10:1   70 EtOH:H₂O 10:1.5 70

The powder diffractogram of Form Compound 1 B-HCl is shown in FIG. 16.

TABLE 9 Representative XRPD peaks of Compound 1 Form B-HCl 2-Theta(degrees) Relative Intensity (%) 8.3 93.7 9.0 8.4 10.9 0.8 11.4 1.4 13.04.9 14.1 19.8 14.8 32.7 15.2 12.6 16.7 23.8 17.8 37.8 18.0 90.0 18.228.6 19.3 19.0 19.5 17.5 19.9 2.7 20.4 9.4 20.6 6.2 21.7 41.2 22.0 22.223.0 100.0 23.6 20.5 23.9 4.0 24.1 3.9 24.5 9.2 24.7 13.0 24.9 31.9 25.222.6 25.7 12.6 26.1 3.3 26.7 4.5 27.1 21.3 27.9 10.6 28.1 18.7 28.5 4.328.7 5.8 29.7 11.1 29.8 14.2 30.1 4.0 30.5 8.2 31.1 30.2 31.5 9.1 32.311.4 32.8 3.8 33.1 9.2 33.4 11.3 33.8 11.1 33.9 10.1 34.1 5.6 34.6 8.534.9 6.4 35.2 10.8 36.0 4.1 36.2 13.2 36.4 4.7 37.2 5.9 37.6 3.7 37.92.2 38.2 7.5 38.5 22.3 38.6 13.8 39.9 10.7

A single crystal of Compound 1 Form B-HCl was determined to possess amonoclinic crystal system, a P2₁/a space group, and the following unitcell dimensions: a=12.57334(5) Å, b=19.68634(5) Å, c=8.39399(5) Å,α=90°, β=90.0554°, and γ=90°.

A sample of Compound Form B-HCl was also evaluated using microscopy.

A DSC curve for Compound 1 Form B-HCl is provided in FIG. 17, and a TGAcurve for a representative sample of Compound 1 Form B-HCl is providedin FIG. 18.

A representative sample of Compound 1 Form B-HCl presented the FTIRspectrum provided in FIG. 19.

Compound 1 Form B-HCl was also analyzed using solid state ¹³C and ¹⁹FNMR. The respective NMR spectra are provided in FIGS. 20 and 21. Severalpeaks found in the ¹³C SSNMR and ¹⁹F SSNMR spectra are described inTables 10 and 11:

TABLE 9 ¹³C SSNMR Peaks for Compound 1 Form B-HCl. Peak No. F1 (ppm) 1176.3 2 168.2 3 148.7 4 143.2 5 138.8 6 131.6 7 129.6 8 129.1 9 126.7 10125.8 11 122.7 12 119.8 13 112.3 14 69.0 15 66.9 16 28.3 17 23.9

TABLE 10 ¹⁹F SSNMR Peaks for Compound 1 Form B-HCl. Peak No. F1 (ppm) 1−55.6 2 −62.0

As a note, in Examples 3A, 3B, and 4A-4C, EtOAC may be used instead of2-MeTHF as the solvent.

Assays for Detecting and Measuring ΔF508-CFTR Potentiation Properties ofCompounds

Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The assay utilizes fluorescent voltage sensing dyes to measure changesin membrane potential using a fluorescent plate reader (e.g., FLIPR III,Molecular Devices, Inc.) as a readout for increase in functionalΔF508-CFTR in NIH 3T3 cells. The driving force for the response is thecreation of a chloride ion gradient in conjunction with channelactivation by a single liquid addition step after the cells havepreviously been treated with compounds and subsequently loaded with avoltage sensing dye.

Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. This HTS assay utilizes fluorescent voltagesensing dyes to measure changes in membrane potential on the FLIPR IIIas a measurement for increase in gating (conductance) of ΔF508 CFTR intemperature-corrected ΔF508 CFTR NIH 3T3 cells. The driving force forthe response is a Cl⁻ ion gradient in conjunction with channelactivation with forskolin in a single liquid addition step using afluorescent plate reader such as FLIPR III after the cells havepreviously been treated with potentiator compounds (or DMSO vehiclecontrol) and subsequently loaded with a redistribution dye.

Solutions

Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH.

An alternative to Bath Solution #1 includes a bath solution where thechloride salts are substituted with gluconate salts.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at ˜20,000/well in 384-wellmatrigel-coated plates and cultured for 2 hrs at 37° C. before culturingat 27° C. for 24 hrs. for the potentiator assay. For the correctionassays, the cells are cultured at 27° C. or 37° C. with and withoutcompounds for 16-24 hours.

Electrophysiological Assays for Assaying ΔF508-CFTR ModulationProperties of Compounds.

1. Ussing Chamber Assaying

Ussing chamber experiments were performed on polarized airway epithelialcells expressing ΔF508-CFTR to further characterize the ΔF508-CFTRmodulators identified in the optical assays. Non-CF and CF airwayepithelia were isolated from bronchial tissue, cultured as previouslydescribed (Galietta, L. J. V., Lantero, S., Gazzolo, A., Sacco, O.,Romano, L., Rossi, G. A., & Zegarra-Moran, O. (1998) In Vitro Cell. Dev.Biol. 34, 478-481), and plated onto Costar® Snapwell™ filters that wereprecoated with NIH3T3-conditioned media. After four days the apicalmedia was removed and the cells were grown at an air liquid interfacefor >14 days prior to use. This resulted in a monolayer of fullydifferentiated columnar cells that were ciliated, features that arecharacteristic of airway epithelia. Non-CF HBE were isolated fromnon-smokers that did not have any known lung disease. CF-HBE wereisolated from patients homozygous for ΔF508-CFTR.

HBE grown on Costar® Snapwell™ cell culture inserts were mounted in anUsing chamber (Physiologic Instruments, Inc., San Diego, Calif.), andthe transepithelial resistance and short-circuit current in the presenceof a basolateral to apical Cl⁻ gradient (I_(SC)) were measured using avoltage-clamp system (Department of Bioengineering, University of Iowa,IA). Briefly, HBE were examined under voltage-clamp recording conditions(V_(hold)=0 mV) at 37° C. The basolateral solution contained (in mM) 145NaCl, 0.83 K₂HPO₄, 3.3 KH₂PO₄, 1.2 MgCl₂, 1.2 CaCl₂, 10 Glucose, 10HEPES (pH adjusted to 7.35 with NaOH) and the apical solution contained(in mM) 145 NaGluconate, 1.2 MgCl₂, 1.2 CaCl₂, 10 glucose, 10 HEPES (pHadjusted to 7.35 with NaOH).

Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. Forskolin (10μM) and all test compounds were added to the apical side of the cellculture inserts. The efficacy of the putative ΔF508-CFTR potentiatorswas compared to that of the known potentiator, genistein.

Patch-Clamp Recordings

Total Cl⁻ current in ΔF508-NIH3T3 cells was monitored using theperforated-patch recording configuration as previously described (Rae,J., Cooper, K., Gates, P., & Watsky, M. (1991) J. Neurosci. Methods 37,15-26). Voltage-clamp recordings were performed at 22° C. using anAxopatch 200B patch-clamp amplifier (Axon Instruments Inc., Foster City,Calif.). The pipette solution contained (in mM) 150 N-methyl-D-glucamine(NMDG)-Cl, 2 MgCl₂, 2 CaCl₂, 10 EGTA, 10 HEPES, and 240 μg/mlamphotericin-B (pH adjusted to 7.35 with HCl). The extracellular mediumcontained (in mM) 150 NMDG-Cl, 2 MgCl₂, 2 CaCl₂, 10 HEPES (pH adjustedto 7.35 with HCl). Pulse generation, data acquisition, and analysis wereperformed using a PC equipped with a Digidata 1320 A/D interface inconjunction with Clampex 8 (Axon Instruments Inc.). To activateΔF508-CFTR, 10 μM forskolin and 20 μM genistein were added to the bathand the current-voltage relation was monitored every 30 sec.

Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in IΔ_(F508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1×pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

Single-Channel Recordings

Gating activity of wt-CFTR and temperature-corrected ΔF508-CFTRexpressed in NIH3T3 cells was observed using excised inside-out membranepatch recordings as previously described (Dalemans, W., Barbry, P.,Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G.,Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature 354, 526-528)using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.).The pipette contained (in mM): 150 NMDG, 150 aspartic acid, 5 CaCl₂, 2MgCl₂, and 10 HEPES (pH adjusted to 7.35 with Tris base). The bathcontained (in mM):150 NMDG-Cl, 2 MgCl₂, 5 EGTA, 10 TES, and 14 Tris base(pH adjusted to 7.35 with HCl). After excision, both wt- and ΔF508-CFTRwere activated by adding 1 mM Mg-ATP, 75 nM of the catalytic subunit ofcAMP-dependent protein kinase (PKA; Promega Corp. Madison, Wis.), and 10mM NaF to inhibit protein phosphatases, which prevented current rundown.The pipette potential was maintained at 80 mV. Channel activity wasanalyzed from membrane patches containing ≦2 active channels. Themaximum number of simultaneous openings determined the number of activechannels during the course of an experiment. To determine thesingle-channel current amplitude, the data recorded from 120 sec ofΔF508-CFTR activity was filtered “off-line” at 100 Hz and then used toconstruct all-point amplitude histograms that were fitted withmultigaussian functions using Bio-Patch Analysis software (Bio-LogicComp. France). The total microscopic current and open probability(P_(o)) were determined from 120 sec of channel activity. The P_(o) wasdetermined using the Bio-Patch software or from the relationshipP_(o)=I/i(N), where I=mean current, i=single-channel current amplitude,and N=number of active channels in patch.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Compound 1 is useful as modulators of ATP binding cassette transporters.The EC₅₀ (μm) of Compound 1 Form A was measured to be less than 2.0 μM.The efficacy of Compound 1 Form A was calculated to be from 100% to 25%.It should be noted that 100% efficacy is the maximum response obtainedwith 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol.

Other Embodiments

All publications and patents referred to in this disclosure areincorporated herein by reference to the same extent as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference. Should themeaning of the terms in any of the patents or publications incorporatedby reference conflict with the meaning of the terms used in thisdisclosure, the meaning of the terms in this disclosure are intended tobe controlling. Furthermore, the foregoing discussion discloses anddescribes merely exemplary embodiments of the present invention. Oneskilled in the art will readily recognize from such discussion and fromthe accompanying drawings and claims, that various changes,modifications and variations can be made therein without departing fromthe spirit and scope of the invention as defined in the followingclaims.

1. A process for producing Compound 1 having solid Form B:

comprising: (a) reacting Compound 2 with the hydrochloride salt ofCompound 3 (3-HCl) in the presence of a coupling agent selected from thegroup consisting of 2-chloro-1,3-dimethyl-2-imidazoliumtetrafluoroborate, HBTU, HCTU, 2-chloro-4,6-dimethoxy-1,3,5-thazine,HATU, HOBT/EDC, and 2-propane phosphonic anhydride

wherein the Form B is characterized by a peak at about 165.3 ppm, a peakat about 145.9 ppm, a peak at about 132.9 ppm , and a peak about 113.4ppm in a ¹³C NMR spectrum.
 2. The process of claim 1, wherein thecoupling of Compound 2 and the hydrochloride salt 3-HCl is performed inthe presence of a base selected from K₂CO₃, Et₃N, N-methylmorpholine(NMM), pyridine or DIEA.
 3. The process of claim 2, wherein the couplingof Compound 2 and the hydrochloride salt 3-HCl is performed in thepresence of pyridine.
 4. The process of claim 1, wherein the coupling ofCompound 2 and the hydrochloride salt 3-HCl is performed in the presenceof a solvent selected from EtOAc, IPAc, THF, MEK, NMP, acetonitrile,DMF, or 2-methyltetrahydrofuran.
 5. The process of claim 4, wherein thecoupling of Compound 2 and the hydrochloride salt 3-HCl is performed inthe presence of a 2-methyltetrahydrofuran.
 6. The process of claim 1,wherein the coupling of Compound 2 and the hydrochloride salt 3-HCl is sperformed at a reaction temperature that is maintained between 15° C.and 70° C.
 7. The process of claim 1, wherein the Form B ischaracterized by a peak at about −56.1 ppm and a peak at about −62.1 ppmin a ¹⁹F NMR spectrum.
 8. The process of claim 7, wherein the couplingof Compound 2 and the hydrochloride salt 3-HCl is performed in thepresence of a base selected from K₂CO₃, Et₃N, N-methylmorpholine (NMM),pyridine or DIEA.
 9. The process of claim 8, wherein the coupling ofCompound 2 and the hydrochloride salt 3-HCl is performed in the presenceof pyridine.
 10. The process of claim 7, wherein the coupling ofCompound 2 and the hydrochloride salt 3-HCl is performed in the presenceof a solvent selected from EtOAc, IPAc, THF, MEK, NMP, acetonitrile,DMF, or 2-methyltetrahydrofuran.
 11. The process of claim 10, whereinthe coupling of Compound 2 and the hydrochloride salt 3-HCl is performedin the presence of 2-methyltetrahydrofuran.
 12. The process of claim 7,wherein the coupling of Compound 2 and the hydrochloride salt 3-HCl is sperformed at a reaction temperature that is maintained between 15° C.and 70° C.
 13. The process of claim 1, wherein the solid Form B ischaracterized by a peak at about 6.7 degrees, a peak at about 10.0degrees, a peak at about 11.2 degrees, a peak at about 13.4 degrees, anda peak at about 24.2 degrees in an X-ray powder diffraction, and whereinthe XRPD peaks are measured on a 2-theta scale.
 14. The process of claim13, wherein the coupling of Compound 2 and the hydrochloride salt 3-HClis performed in the presence of a base selected from K₂CO₃, Et₃N,N-methylmorpholine (NMM), pyridine or DIEA.
 15. The process of claim 14,wherein the coupling of Compound 2 and the hydrochloride salt 3-HCl isperformed in the presence of pyridine.
 16. The process of claim 13,wherein the coupling of Compound 2 and the hydrochloride salt 3-HCl isperformed in the presence of a solvent selected from EtOAc, IPAc, THF,MEK, NMP, acetonitrile, DMF, or 2-methyltetrahydrofuran.
 17. The processof claim 16, wherein the coupling of Compound 2 and the hydrochloridesalt 3-HCl is performed in the presence of 2-methyltetrahydrofuran. 18.The process of claim 13, wherein the coupling of Compound 2 and thehydrochloride salt 3-HCl is s performed at a reaction temperature thatis maintained between 15° C. and 70° C.
 19. The process of claim 1,wherein the solid Form B is characterized by a single crystal whichpossesses a monoclinic crystal system, a P21/c space group, and thefollowing unit cell dimensions: a =13.5429(4) 521 ; b =13.4557(4) 521 ;c =12.0592(4) 521 ; α=90°; β=101.193°; and γ=90°.
 20. The process ofclaim 19, wherein the coupling of Compound 2 and the hydrochloride salt3-HCl is performed in the presence of a base selected from K₂CO₃, Et₃N,N-methylmorpholine (NMM), pyridine or DIEA.
 21. The process of claim 20,wherein the coupling of Compound 2 and the hydrochloride salt 3-HCl isperformed in the presence of pyridine.
 22. The process of claim 19,wherein the coupling of Compound 2 and the hydrochloride salt 3-HCl isperformed in the presence of a solvent selected from EtOAc, IPAc, THF,MEK, NMP, acetonitrile, DMF, or 2-methyltetrahydrofuran.
 23. The processof claim 22, wherein the coupling of Compound 2 and the hydrochloridesalt 3-HCl is performed in the presence of 2-methyltetrahydrofuran. 24.The process of claim 9, wherein the coupling of Compound 2 and thehydrochloride salt 3-HCl is s performed at a reaction temperature thatis maintained between 15° C. and 70° C.